Mesenchymal precursor cell

ABSTRACT

A method of enriching mesenchymal precursor cells including the step of enriching for cells based on at least two markers. The markers may be either i) the presence of markers specific for mesenchymal precursor cells, ii) the absence of markers specific for differentiated mesenchymal cells, or iii) expression levels of markers specific for mesenchymal precursor cells. The method may include a first solid phase sorting step utilising MACS recognising expression of the antigen to the STRO-1 Mab, followed by a second sorting step utilising two colour FACS to screen for the presence of high level STRO-1 antigen expression as well as the expression of VCAM-1.

This invention relates to the enrichment of mesenchymal precursor cellsusing a combination of cell surface markers, and to a cell population ofmesenchymal precursor cells.

Mesenchymal cells are derived from a number of tissues and act as thesupportive structure for other cell types. Bone marrow for instance ismade of both haematopoietic and mesenchymal derived cells. Themesenchymal cells include endothelial cells that form the sinuses andadvetitial reticular cells that have characteristics consistent withadipocytes, fibroblasts and muscle cells.

It is believed that certain mesenchymal precursor cells (MPCs) areresponsible for the formation of mesenchymal cells. In the bone MPCs arethe formative pluripotent blast cells that are believed to be capable ofdifferentiating into any of the specific types of connective tissues(ie. the tissue of the body that support the specialised elements,particularly adipose, areolar, osseous, cartilaginous, elastic andfibrous connective tissues) depending upon the various environmentalinfluences.

Purification or at least enrichment of MPCs is desirable for a varietyof therapeutic reasons. The reasons include regeneration of missing ordamaged skeletal tissue, enhancing the implantation of various plasticor metal prosthetic devices through the attachment of the isolated andculturally expanded marrow derived mesenchymal cells onto the poroussurfaces of the prosthetic devices, which upon activation and subsequentdifferentiation of marrow-derived mesenchymal cells produce naturalosseous bridges.

Composite grafts of cultured mesenchymal cells might be used to augmentthe rate of haematopoietic cell reserve during bone marrowtransplantation.

A class of defects that may be repaired by cultured marrow-derivedmesenchymal cells expanded from the MPCs of the present invention is theclass of large skeletal defects in bone caused by injury or produced bythe removal of large sections of bone infected with tumour. Under normalcircumstances this type of defect does not heal and creates nonunion ofthe bone. This type of defect may be treated by implanting culturedmesenchymal cells contained in calcium phosphate ceramic vehicles intothe defect site.

A second class of defect that may be repaired by cultured marrow-derivedmesenchymal cells expanded from the MPCs of the present invention, isthe damaged articular cartilage generated by trauma or by diseases suchas osteoarthritis and rheumatoid arthritis. Under normal circumstances,damage to articular cartilage does not heal except in very youngindividuals where the underlying bone is also damaged so that a bloodywound is created. It is projected by the present invention that thistype of defect can be treated by implanting cultured marrow derivedmesenchymal cells into the defect. The cells will be formatted incarriers which will hold the cells in the defect and present them in amanner (round cell morphology) that they differentiate intochondrocytes.

It is not clearly understood why composite grafts of culturedmesenchymal cells and ceramic induce recruitment of haematopoietic stemcells and other marrow elements, however, the fact that this does occurallows for the use of these grafts in a way to sequester haematopoieticstem cells and generate a haematopoietic stem cell reservoir. Thereservoir of haematopoietic stem cells can then be used in clinicalapplications such as marrow transplantation as an alternative method forharvesting haematopoietic stem cells.

Another potential use for purified cells is as a means of gene therapy,by the introduction of exogenous nucleic acids for the expression oftherapeutic substances in the bone marrow—see U.S. Pat. No. 5,591,625 byGerson et al.

A purified source of MPCs is desirable for a number of reasons. Onemajor reason is that if there is a mixed population, MPCs will respondto signals elicited by other cells to behave in a manner that might notbe desired. Thus, for example, a contaminating cell might express acytokine that evokes differentiation into adipose tissue, whereas onemay require the cells for bone formation, in which case the usefulnessof the MPCs is somewhat limited. Additionally for a reason similar tothat given above, purified progenitor cells tend to be easier to handleand manage than less purified cells.

There have been many attempts at purifying or significantly enrichingMPCs, however significant enrichment has until the present invention notbeen achieved. In contrast to the haematopoietic system, in which stemcells can be physically separated based upon differences in theirexpression of cell surface markers, the cell surface antigenic phenotypeof MPCs remains relatively poorly defined. A further problem ofpurification of MPCs is a result of the physical association betweenmesenchymal cells and other cell types.

The bone and bone marrow (BM) tissues contain a phenotypically diversepopulation of stromal cell lineages that are currently thought to arisefrom a rare and primitive population of multi-potential mesenchymalprecursor cells (MPC) [Owen, 1985; Owen and Friedenstein, 1988]. Bonemarrow MPC can be readily measured by their ability to form adherentclonogenic clusters composed of fibroblastic-like cells (CFU-F:colony-forming-unit-fibroblast) in short-term liquid culture[Friedenstein et al, 1970; Castro-Malaspina et al, 1980]. In vitrostudies have documented variations in the morphology and proliferativecapacity of different BM MPC clones [Friedenstein et al, 1970; 1976;Castro-Malaspina et al, 1980; Owen et al, 1987; Bennett et al, 1991;Simmons and Gronthos, 1991]. The heterogeneous nature of the BM MPCpopulation was further demonstrated iii studies where culture expandedMPC clones displayed different developmental potentials in the presenceof glucocorticoids or when transferred into ectopic sites in vivo[Friedenstein et al, 1980; Owen et al, 1987; Bennett et al, 1991].Collectively, these studies support the concept of a stromal cellhierarchy of cellular differentiation by analogy with the haematopoieticsystem.

Given the extensive literature regarding the characterisation ofhaematopoietic stem cells and their progeny there has been littleprogress towards the identification of the various elements whichconstitute the bone marrow stromal precursor compartment. This is due inpart to the low incidence of MPC in aspirates of marrow (0.05% to0.001%) [Castro-Malaspina et al 1980; Simmons and Torok-Storb, 1991a;1991b; Fella et al, 1993; Waller et al, 19954 and because of the paucityof antibody reagents that allow for the precise identification andisolation of the MPC population. Stromal precursor cells have beenpartially enriched from bone marrow aspirates through their binding todifferent lectins such as soya bean agglutinin and wheat germ agglutininor by using a negative immunoselection process based on their lack ofexpression of various cell surface antigens restricted to the myeloid,erythroid and lymphoid cell lineages [Simmons and Torok-Storb 1991a;1991b; Simmons et al, 1994; Rickard et al, 1996]. However, theinefficiency of these selection strategies has resulted in the presenceof contaminating populations of accessory cells and haematopoieticprogenitor cells. Moreover, a major difficulty in using techniques suchas fluorescence activated cell sorting (FACS) to positively select forpure populations of MPC is that they share many common antigens with HSCincluding early developmental markers such as the human CD34 antigen andthe murine stem cell antigen-1.

Recent advances in the study of human stromal stem cell biology havebeen attributed to the development of novel monoclonal antibodies (Mabs)which recognise antigens on BM MPC that are correspondingly not reactivewith haematopoietic progenitors. We have previously described amonoclonal antibody, STRO-1 which identifies an as yet unidentified 60kDa cell surface antigen expressed on all assayable MPC in aspirates ofadult human BM [Simmons and Torok-Storb, 1991a]. The majority of theSTRO-1⁺ bone marrow mononuclear cells (BMMNC) (approximately 90%) havebeen identified as late stage glycophorin A⁺ erythroblasts. The MPCpopulation are restricted to the minor population of STRO-1⁺ cells whichlack glycophorin A [Simmons and Torok-Strob, 1991a]. Importantly, STRO-1demonstrates no detectable binding to haematopoietic progenitors(CFU-GM, BFU-E, BFU-Meg, CFU-GEMM) nor to their precursors (pre-CFU)[Simmons and Torok-Storb, 1991a; Gronthos and Simmons, unpublishedobservations].

A systematic examination of the immunophenotype of MPC derived fromadult human BM has previously been performed using two-color FACSanalysis [Simmons et al, 1994]. A number of antigens were shown to becoexpressed with STRO-1 by essentially all BM MPC. These included theendopeptidases CD10 and CD13 and the adhesion molecules Thy-1 (CDw90),VCAM-1(CD106) and various members of the β1 (CD29) integrin family[Simmons et al, 1994]. This is in accord with the data of Terstappen andcolleagues regarding the antigenic phenotype of human foetal BM MPC[Waller et al, 1995].

SUMMARY OF THE INVENTION

This invention arises from the finding that enrichment of mesenchymalprecursor cells is greatly enhanced by the use of two markers specificfor mesenchymal cells, that can be used to recognise early cells. Tothis end it will be appreciated that MPCs arc early cells that aresubstantially at a pre-expansion stage of development and hence areprecursors to mesenchymal stem cells in which a significant number ofthe population have expanded and are therefore incapable of furtherexpansion. Thus, MPCs are cells that have yet to differentiate to fullycommitted mesenchymal cells. These cells need not however be stem cellsin a strict sense, in that they are necessarily able to differentiateinto all types of mesenchymal cells. There is a benefit in having anenriched pool of MPCs that are able to differentiate into bone formingcells only, in that these precursor cells have a greater proliferationpotential. In particular in accordance with the present inventionbecause the proportions of MPCs in the 1 harvested population is large,the extent to which the population can be expanded is greatly enhanced.Additionally according to aspects of this invention it has beendiscovered that MPCs are present in the perivascular compartment and areable to be purified from a range of tissues.

Aspects of the present invention provides an enrichment several ordersof magnitude better than the best method known to the inventors beforethe present invention. The inventors have shown that an enrichedpopulation in which up to 50% of the MPCs can form colonies of ten ormore cells can be achieved using the present invention. In contrast, thecitations indicate that the best method known up until now has onlyachieved an enrichment of up to 0.01% cells capable of forming colonies.It is to be noted that as discussed herein the presence of MPCs is basedupon their colonigenic capacity, as determined by the presence ofcolonies of ten or more cells in liquid culture seeded with single cellsafter having been grown for 14 days.

In a broad form of a first aspect the invention could be said to residein a method of enriching mesenchymal precursor cells (MPCs) the methodincluding the steps of enriching for cells based on at least twomarkers, said markers being either the presence of, or expression levelsof markers specific for mesenchymal precursor cells on the one hand, orabsence of marker or levels of expression specific for differentiatedmesenchymal cells on the other hand.

The preferred source of material for enrichment is bone marrow, and thusin a one form the method is limited to the enrichment of bone marrowderived mesenchymal stem cells. It is also likely that the method ofthis first aspect of the invention might be used to enrich stromal stemcells from other sources such as blood, epidermis and hair follicles. Itis proposed that mesenchymal precursor cells isolated from, for example,skin should have the same potential as those cells isolated from bonemarrow. An advantage in isolating cells from skin is that the harvestingis far less invasive than the harvesting of a sample of bone marrow.

It is anticipated that a proportion of the population purified will bestem cells, however, it is not yet known how to separate these stemcells from the MPC population. It has been observed however that asubpopulation has a much greater capacity to divide than others, andperhaps this subpopulation represents the stem cells. It is estimatedthat approximately 10 to 20% of the MPCs isolated by the illustratedmethod of this invention are stem cells.

It is preferred that a significant proportion of the MPCs are capable ofdifferentiation into at least two committed cell types selected from thegroup including but not limited to adipose, areolar, osseous,cartilaginous, elastic and fibrous connective.

It has been found that it is possible to purify MPCs by the above methodto a degree where these cells are present in a purified population ofwhich 50% of the MPCs can form colonies of ten or more cells. Thereforethe method may result in a cell population in which at least 1% of thecells are MPCs that are colony forming, preferably at least 5% of thecells are MPCs that are colony forming, more preferably at least 10% ofthe cells are MPCs that are colony forming, and most preferably at least40% of the cells are MPCs that are colony forming.

The nearest known purification is that by Pittenger et al. (Science 284;143-147) where cells had been enriched using a Percoll gradient. Theseworkers were only able to get colony forming units from 0.001-0.01% ofcells. The present technique therefore results in a very significantenrichment when compared to these attempts.

The present invention is also to be contrasted to the enrichedpopulations described by Caplan et al. in U.S. Pat. No. 5,837,539 whodescribes a method for the isolation, purification and culture expansionof mesenchymal stem cells which is said to give compositions havinggreater than 95% human mesenchymal stem cells. It is to be noted thatthe figure of 95% relates to populations of expanded mesenchymal stemcells, and is likely to reflect a lower number of colony forming unitsbecause the cells are at least partially expanded. Thus, Caplan startswith a population of BM cells comprising about 1 in 1000 MPCs andexpands the population and then purifies the at least partially expandedpopulation. In contrast the present invention can result in a populationof about 1 in 2 cells that are able to form colonies of at least 10MSCs.

Preferably the method includes enriching by selecting for the positiveexpression of at least one marker and more preferably both markers areselected for positive expression. These markers are most convenientlycell surface markers. The markers might be selected from a group ofsurface markers specific for MPC including but not limited to LFA-3,THY-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, CD49b/CD29,CD49c/CD29, CD49d/CD29, CD29, CD18, CD61, 6-19, thrombomodulin, CD10,CD13, SCF, and the antigen recognised by STRO-1.

Reagents suitable for use in labelling these markers can be found inTable 4.

The marker might be absence of various surface markers indicative ofcommitment, such as CBFA-1, collagen type II, PPARγ2, glycophorin A.

In one preferred form at least one of the markers is the antigenrecognised by STRO-1, and in particular the high level of expression ofthat antigen.

In another preferred form at least one of the markers is VCAM-1.

In one very specific form the two markers are the antigen recognised bySTRO-1 and VCAM-1.

The specificity of the markers used in this process is not absolute.Thus even the most preferred markers occur on cell types other thanmesenchymal cells, however their expression on the cell surfaces ofother cell types is limited.

It will be understood that recognition of cells carrying the cellsurface markers that form the basis of the separation can be effected bya number of different methods, however, all of these methods rely uponbinding a binding agent to the marker concerned followed by a separationof those that exhibit binding, being either high level binding, or lowlevel binding or no binding. The most convenient binding agents areantibodies or antibody based molecules, preferably being monoclonalantibodies or based on monoclonal antibodies because of the specificityof these latter agents. Antibodies can be used for both steps, howeverother agents might also be used, thus ligands for these markers may alsobe employed to enrich for cells carrying them, or lacking them.

The antibodies may be attached to a solid support to allow for a crudeseparation. The separation techniques should maximise the retention ofviability of the fraction to be collected. Various techniques ofdifferent efficacy may be employed to obtain relatively crudeseparations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS.

The method might include the step of making a first partially enrichedpool of cells by enriching for the expression of a first of the markers,and then the step of enriching for expression of the second of themarkers from the partially enriched pool of cells.

It is preferred that the method comprises a first step being a solidphase sorting step, based on recognition of one or more of the markers.The solid phase sorting step of the illustrated embodiment utilises MACSrecognising high level expression of STRO-1. This then gives an enrichedpool with greater numbers of cells than if a high accuracy sort was usedas a first step. If for example FACS is used first, many of the MPCs arerejected because of their association with other cells. A second sortingstep can then follow using an accurate separation method. This secondsorting step might involve the use of two or more markers. Thus in theillustrated embodiment two colour FACS is used to recognise high levelexpression of the antigen recognised by STRO-1 as wells as theexpression of VCAM-1. The windows used for sorting in the second stepcan be more advantageously adjusted because the starting population isalready partially enriched.

It will be understood that the invention is not limited to theenrichment of cells by their expression of only two markers and it maybe preferred to enrich based on the expression of three or more markers.

The method might also include the harvesting of a source of the stemcells before the first enrichment step, which in the most preferredsource comprises the step of harvesting bone marrow cells, using knowntechniques.

The preferred source of such cells is human, however, it is expectedthat the invention is also applicable to animals, and these mightinclude domestic animals or animals that might be used for sport.

In a broad form of a second aspect the invention could be said to residein an enriched population of mesenchymal precursor cells as purified bya method according to the first aspect of the invention.

It has been found that it is possible to purify MPCs to a degree wherethe purified population contains 50% of these cells that are capable offorming colonies of 10 or more cells.

In a broad form of a third aspect the invention could also be said toreside in a cell population in which at least 1% of the cells are MPCsthat are colony forming, preferably at least 5% of the cells are MPCsthat are colony forming, more preferably at least 10% of the cells areMPCs that are colony forming, and most preferably at least 40% of thecells are MPCs that are colony forming.

The cells of the enriched population preferably carry at least twomarkers selected from a group of surface markers specific formesenchymal precursor cells including LFA-3, THY-1, antigen identifiedby STRO-1, VCAM-1, ICAM-1, PECAM-1, P-selectin, L-selectin, CD49b/CD29,CD49c/CD29, CD49d/CD29, CD29, CD18, CD61, 6-19, thrombomodulin, CD10,CD13 and SCF. Most preferably the cells carry the antigen identified bySTRO-1 and VCAM-1.

It will also be understood that in a fourth aspect the inventionencompasses a composition including the purified MPCs or a compositionmade from the purified MPCs.

The purified population of the second or third aspects of the invention,or the composition of the fourth aspect of the invention might be usedin the formation and repair of bones, and as such a combination of MPCsas well as a suitable support may be introduced into a site requiringbone formation. Thus, for example, skeletal defects caused by boneinjury or the removal of sections of bone infected with tumour may berepaired by implanting cultured MSCs contained in calcium phosphateceramic vehicles into the defect site. For appropriate methods andtechniques see Caplan et al. in U.S. Pat. No. 5,226,914 and U.S. Pat.No. 5,837,539, both of which use cruder preparations of stem cells.

In addition, the enriched population or composition may be used toassist in anchoring prosthetic devices. Thus, the surface of aprosthetic device such as those used in hip, knee and shoulderreplacement, may be coated with the enriched MPCs prior to implantation.The MPCs may then differentiate into osteogenic cells to thereby speedup the process of bony ingrowth and incorporation of the prostheticdevice (see Caplan et al. in U.S. Pat. No. 5,226,914 and U.S. Pat. No.5,837,539).

The enriched population or composition might also be used in genetherapy so that, for example, an enriched population may have exogenousnucleic acid transformed into it and then such a population may beintroduced into the body of the patient to treat a disease or condition.Alternatively it might be used for the release of therapeutics. Forappropriate techniques we refer to U.S. Pat. No. 5591625 by Gerson etal. which uses cruder preparations of stem cells.

Alternatively the enriched population or composition may be used toaugment bone marrow transplantation, wherein the composition containingpurified MSCs can be injected into a patient undergoing marrowtransplantation prior to the introduction of the whole marrow. In thisway the rate of haemopoiesis may be increased, particularly followingradiation or chemotherapy. The composition might also encompass amixture of MPCs and haematopoietic cells which may be useful inradiotherapy or chemotherapy.

In a first form of a fifth aspect the invention might be said to residein a method of enriching for mesenchymal precursor cells (MPCs), themethod including the step of preparing a single cell suspension from avascularised source tissue and the step of enriching based on thepresence of an early perivascular cell marker.

In a second form of the fifth aspect the invention might be said toreside in a method of enriching for mesenchymal precursor cells, themethod including the step of preparing a single cell suspension from a,non-bone marrow, vascularised source tissue and separating the tissueinto separate cells and the step of enriching based one of the presenceor level of one or more early developmental markers and the absence ofone or more surface markers indicative of commitment.

In a third form of the fifth aspect the invention might be said toreside in a method of enriching for mesenchymal precursor cells (MPCs),the method including the step of preparing a single cell suspension froma vascularised source tissue and the step of enriching based on thepresence of markers expressed in the vascularized tissue byperi-vascular cells.

In a sixth aspect the invention might be said to reside in an enrichedpopulation of cells enriched for mesenchymal precursor cells (MPCs) saidMPCs having a phenotype of 3G5, MUC18, VCAM-1, STRO-1^(bri) and α smoothmuscle actin.

In a first form of a seventh aspect the invention might be said toreside in an isolated mesenchymal precursor cells (MPCs) said MPCshaving a phenotype of 3G5, MUC18, VCAM-1, STRO-1^(bri) and α smoothmuscle actin.

In a second form of the seventh aspect the invention might be said toreside in an isolated mammalian cell that is multipotent and that ispositive for the surface marker 3G5.

In a third form of the seventh aspect the invention might be said toreside in a mesenchymal precursor cell (MPC), capable of forming aclonogenic colony and differentiating to three or more mesenchymaltissue types, isolated from a tissue of the group comprising, but notlimited to, adipose tissue, teeth, dental pulp, skin, liver, kidney,heart, retina, brain, hair follicles, intestine, lung, spleen, lymphnode, thymus, pancreas, bone, ligament, bone marrow, tendon, andskeletal muscle, and which is positive for the surface marker STRO-1.

In a fourth form of the seventh aspect the invention might be said toreside in an unexpended population of cells enriched for mesenchymalprecursor cells (MPCs), capable of forming a clonogenic colony anddifferentiating to three or more mesenchymal tissue types, said MPCsco-expressing the surface markers MUC18/CD146 and alpha-smooth muscleactin.

In an eighth aspect the invention might be said to reside in adifferentiated progeny cell arising from the third aspect of theinvention preferably wherein the progeny cell is at least an osteoblast,odontoblast, dentin-producing, chondrocyte, tendon, ligament, cartilage,adipocyte, fibroblast, marrow stroma, osteoclast- andhaematopoietic-supportive stroma, cardiac muscle, smooth muscle,skeletal muscle, pericyte, vascular, epithelial, glial, neuronal,astrocyte or oligodendrocyte cell.

Several aspects of present invention relates to mesenchymal precursorcells, in particular those that may be present in the perivascularcompartment of vascularised tissue. Such mesenchymal cells may beidentified by the presence of the 3G5 surface marker, and perhapsadditionally or separately by other early developmental markers such asCD146 (MUC18), VCAM-1 and STRO-1.

Precursor cells are early cells that are substantially at apre-expansion stage of development. These are cells that have yet todifferentiate to fully committed cells, however they need not be stemcells in a strict sense, in that they are necessarily able todifferentiate into all types of cells. Partially differentiatedprecursor cells have a benefit in that they have a greater proliferativepotential than stem cells.

The present precursor cells are somewhat differentiated in that they arecommitted to mesenchymal tissue, as opposed, for example, tohaematopoietic tissues. It is evident from the data produced that theMPCs that have been isolated lack markers associated with haematopoieticcells such as CD34, and additionally their differentiation potentialdoes not extend to haematopoietic lines. Additionally they need notnecessarily have the potential to differentiate into all mesenchymalcell type, rather, they may be able to differentiate into one, two threeor more cell types.

It is anticipated that these precursor cell harvested from the tissuesconcerned may be useful for regenerating tissue for cells types fromwhich they have been sourced. Thus precursor cells isolated from heartmay be reintroduced to regenerate heart tissue, however their potentialneed not be so limited, precursor cells isolated from one tissue typemight be useful for regenerating tissue in another tissue type. Themicroenvironment in which an undifferentiated cell finds itself is knownto exert an influence on the route of differentiation and therefore thereintroduction need not necessarily be tissue specific.

The data presented show that MPCs have been harvested and thenre-introduced to produce bone and bone marrow and dentin and pulprespectively, in addition aterioles, cord like structures, have beenproduced after ex vivo expansion of isolated MPCs.

It is anticipated that a wide range of cells might be produced based ongene expression of markers characteristic for certain cell types. It isthus anticipated that under appropriate culture conditions the range ofcell types that can be generated from the perivascular MPCs of thepresent invention include but are not limited to the following,osteoblast, odontoblast, dentin-producing, chondrocyte, tendon,ligament, cartilage, adipocyte, fibroblast, marrow stroma, osteoclast-and haematopoietic-supportive stroma, cardiac muscle, smooth muscle,skeletal muscle, pericyte, vascular, epithelial, glial, neuronal,astrocyte or oligodendrocyte cell.

One of the benefits of the finding that MPCs can be isolated fromperivascular cells is that this greatly expands the range of sourcetissues from which MPCs can be isolated or enriched and there is nolonger an effective restriction on the source of MPCs to bone marrow.The tissues from which these MPCs have been isolated in theexemplifications of this invention are human bone marrow, dental pulpcells, adipose tissue and skin. In addition in situ staining andhistological studies have identified that MPC are present in theperivascular compartment of spleen, pancreas, brain, kidney, liver andheart. Given this wide and diverse range of tissue types whereperivascular MPCs are present, it is proposed that MPC will also bepresent from an even wider range of tissue which may include, adiposetissue, teeth, dental pulp, skin, liver, kidney, heart, retina, brain,hair follicles, intestine, lung, spleen, lymph node, thymus, pancreas,bone, ligament, bone marrow, tendon, and skeletal muscle.

These precursor cells of the present invention are distinguished fromother known MPCs in that they are positive for 3G5 or perhaps that theycarry another perivascular markers. They can be isolated by enrichingfor an early developmental surface marker present on perivascular cells,in particular the presence of one or more of CD146(MUC18), VCAM-1 andalternatively or additionally high level expression of the markerrecognised by the monoclonal antibody STRO-1. Alternatively oradditionally enrichment may be carried out using 3G5.

Markers associated with perivascular cells may also be present on theMPCs, for example alpha smooth muscle actin (αSMA).

Other early developmental markers associated with MPCs may also bepresent. These may include but are not necessarily limited to the groupconsisting of THY-1, VCAM-1, ICAM-1, PECAM-1, CD49a/CD49b/CD29,CD49c/CD29, CD49d/CD29, CD29, CD61, integrin beta 5, 6-19,thrombomodulin, CD10, CD13, SCF, STRO-1bri, PDGF-R, EGF-R, IGF1-R,NGF-R, FGF-R, Leptin-R (STRO-2). Positive expression of one or more ofthese markers may be used in methods of enriching for MPCs from sourcetissue.

The MPCs of the present invention may also be characterised by theabsence of markers present in differentiated tissue, and enrichment maybe based on the absence of such markers.

Similarly it is preferred that the enriched cell populations are not ofhaematopoietic origin and thus it is preferred that these cells are notpresent. Markers characteristically identified as not present includebut are not limited to CD34, CD45 and glycophorin A. Additional othermarkers for this purpose might include CD20 and CD19 (B lymphocytemarkers), CD117 (c-kit oncoprotein) present on haematopoietic stem cellsand angioblasts, CD14 (macrophage), CD3 and CD4 (T cells).

It may be desirable to use the relatively quiescent, directly enrichedor isolated perivascular MCPs. Alternatively it has been discovered thatexpansion of the enriched population can be carried out and have thebeneficial effect of resulting in much greater numbers of cells. Thoeffect of expansion of the directly enriched pool of cells is, however,that some differentiation of the initial MCPs will occur. Expansion overa 5 week period might result in an increase of 10³ fold. Other periodsmight be chosen to expand the population to between 10² to 10⁵ fold.This potential might be directed by culturing them is media containingcytokines and other factors directing the differentiation to aparticular tissue type for example PDGF and VEGF forming smooth musclealpha cords. These could then be introduce into a tissue with, forexample, an insult to assist with repair. Alternatively it may bedesired after expansion to reselect cells on the basis of an earlydevelopmental marker, that might be STRO-1^(bri) to increase theproportion of MPCs in the population.

It is found that an essentially pure population of MCPs is not necessaryto provide for formation of differentiated cells to form desired tissuestructures. The enriched population may have levels of MCPs of greaterthan about 0.001, 0.01, 0.02, 0.05, 0.1, 0.2, 0.5 or 1% or higher as aproportion of total cell numbers in the enriched population. This orderof enrichment can be achieved by the use of a single marker forselection of the enriched MCP population. This is particularly so wherethe source tissue has an inherently high level of perivascular MCPs. Itis found that considerably more 3G5 pos MCPs are present in certaintissue, for example dental pulp, than in bone marrow. Thus in bonemarrow 3G5 positive MPCs constitute about 15% of MPC based on STR1^(bri)colony forming cells, whereas in dental pulp that are found toconstitute 65% and greater than 90% in fat and skin tissues. Expansionof the population and then re-enrichment using a single marker coungresult in higher leves of MPCs, perhaps levels greater than about 0.1,0.5, 1, 2, 5 or 10%

Whilst it is considered desirable that a substantial proportion andpreferably a majority of precursor cells are perivascular MPCs, it isnot considered essential for certain forms of the invention forperivascular MPCs to be the sole precursor cell form.

Other forms of precursors may also be present without unduly interferingwith the capacity of the perivascular MPCs to undergo the desireddifferentiation. Such other forms may include haematopoietic precursorsor non-perivascular MPCs, perhaps being negative for 3G5.

Certain forms of the present invention provide perivascular MPCssubstantially free of endothelial cells. In that context substantiallyfree might be considered to be less than about 5, 2, 1, or 0.1%endothelial cells. Alternatively the context might be an assessment thatthe enriched population is von Willebrand Factor negative.

It will be understood that recognition of cells carrying the cellsurface markers that form the basis of the separation can be effected bya number of different methods, however, all of these methods rely uponbinding a binding agent to the marker concerned followed by a separationof those that exhibit binding, being either high level binding, or lowlevel binding or no binding. The most convenient binding agents areantibodies or antibody based molecules, preferably being monoclonalantibodies or based on monoclonal antibodies because of the specificityof these latter agents. Antibodies can be used for both steps, howeverother agents might also be used, thus ligands for these markers may alsobe employed to enrich for cells carrying them, or lacking them.

The antibodies may be attached to a solid support to allow for a crudeseparation. The separation techniques should maximise the retention ofviability of the fraction to be collected. Various techniques ofdifferent efficacy may be employed to obtain relatively crudeseparations. The particular technique employed will depend uponefficiency of separation, associated cytotoxicity, ease and speed ofperformance, and necessity for sophisticated equipment and/or technicalskill. Procedures for separation may include, but are not limited to,magnetic separation, using antibody-coated magnetic beads, affinitychromatography and “panning” with antibody attached to a solid matrix.Techniques providing accurate separation include but are not limited toFACS.

It is in the context of these methods that a cell be either negative orpositive. The positive cells may either be low (lo) or a hi (bright)expresser depending on the degree to which the marker is present on thecell surface, the terms relate to intensity of fluorescence or othercolor used in the color sorting process of the cells. The distinction oflo and bri will be understood in the context of the marker used on aparticular cell population being sorted.

The method of enriching for perivascular MPCs might include the step ofmaking a first partially enriched pool of cells by enriching for theexpression of a first of the markers, and then the step of enriching forexpression of the second of the markers from the partially enriched poolof cells.

It is preferred that the method comprises a first step being a solidphase sorting step, based on recognition of one or more of the markers.The solid phase sorting step of the illustrated embodiment utilises MACSrecognising high level expression of STRO-1. This then gives an enrichedpool with greater numbers of cells than if a high accuracy sort was usedas a first step. If for example FACS is used first, many of theprecursor cells are rejected because of their association with othercells. A second sorting step can then follow using an accurateseparation method. This second sorting step might involve the use of twoor more markers. Thus in the illustrated embodiment two colour FACS isused to recognise high level expression of the antigen recognised bySTRO-1 as wells as the expression of CD146. The windows used for sortingin the second step can be more advantageously adjusted because thestarting population is already partially enriched.

The method of enriching for perivascular MPCs might also include theharvesting of a source of the stem cells before the first enrichmentstep using known techniques. Thus the tissue will be surgically removed.Cells comprising the source tissue will then be separated into a socalled single cells suspension. This separation may be achieved byphysical and or enzymatic means.

The preferred source of such perivascular MPCs is human, however, it isexpected that the invention is also applicable to animals, and thesemight include agricultural animals such as cows, sheep, pigs and thelike, domestic animals such as dogs, laboratory animals such as mice,rats, hamsters, and rabbits or animals that might be used for sport suchas horses.

In a further form the invention might be said to reside a method ofgeneration tissue in a mammal comprising the step of enriching apopulation of precursor cells as in the first aspect of the invention,and introducing the enriched population into the mammal, and allowingthe enriched population to generate the tissue in the mammal.

Another potential use for enriched cells of the present invention is asa means of gene therapy, by the introduction of exogenous nucleic acidsfor expression of therapeutic substances in the tissue types concerned.

In the context of the present invention the term isolated cell may meanthat perivascular MPCs comprise at least 30, 40, 50, 60, 70, 80, or 95%of total cells of the population in which they are present.

FIGURE LEGENDS

FIG. 1 The frequency histogram represents the immunofluorescenceanalysis by FACS of BMMNC isolated by MACS on the basis of STRO-1 (FITC)expression: STRO-1^(dull) cell fraction (A); STRO-1^(intermediate) cellfraction (B); STRO-1^(bright) cell fraction (C); The histogram is basedon 10⁴ events collected as list mode data.

FIG. 2 Dual-colour flow cytometric analysis of VCAM-1 (PE) expression bySTRO-1⁺ (FITC) BMMNC isolated by MACS. The dot plot histogram represents5×10⁴ events collected as listmode data. STRO-1^(bright)/VCAM-1⁺ cellswere sorted by FACS (rectangle), which represented approximately 0.1% ofthe total BMMNC population (A). The incidence of clonogenic cells (B)colonies (>50 cells) and (C) colonies+clusters (>10<50 cells) based onSTRO-1^(bright)/VCAM-1³⁰ expression. The frequency of clonogenic cellswas determined by limiting dilution analysis (24 replicates per cellconcentration) employing Poisson distribution analysis.

FIG. 3 Characterization of BM MPC. (A) Light microscopic examination ofthe freshly sorted cells revealed a homogenous population of large cellswith heterochromatic nuclei and prominent mucleoli, a granular cytoplasmand numerous blel-like projections of the cell membrane (magnified 40×).(B) Transmission electron micrograph of STRO-1^(bright)/VCAM-1⁺ sortedcells isolated directly from BM (magnified 1000×). (C)Immunohistological staining of cytospin preparations of the sortedSTRO-1^(bright)/VCAM-1⁺ BMMNC showing intense staining of most cellswith anti-collagen type I antibody, (magnified 40×). (D) Lightmicroscopic view of a purified STRO-1^(bright)/VCAM-1⁺, allowed toadhere to fibronectin-coated culture adopts a stellate, fibroblastoidmorphology.

FIG. 4 Characterization of BM MPC. Dual-colour flow cytometric analysisof Ki67 (FITC) expression by STRO-1⁺ (PE) BMMNC isolated by MACS. Thedot plot histogram represents 5×10⁴ events collected as listmode data(B). Telomerase activity in sorted cells populations was examined usinga modified TRAP assay (C). TRAP products derived from CHAPS extracts ofnon-denatured (−) and denatured (+) total bone marrow (lanes 1 and 2),Total STRO-1 [MACS-selected] (lanes 2 and 3). STRO-1^(bright)/VCAM-1⁺cells sorted fraction (lanes 4 and 5), cultured. STRO-1^(bright)/VCAM-1⁺cells (lanes 6 and 7) and CD34⁺-sorted cells TRAP products were resolvedon a 12% polyacrylamide gel, stained with SYBR green fluorescent dye,and visualised using a fluorescence scanning system.

FIG. 5 A total of 44 CFU-F colonies derived from two BM samples wereanalysed for their cumulative production of cells. A marked variation inproliferative capacity between individual MPC is evident. The majorityof clones (36/44; 82%) exhibited only modertate growth potential whichdid not persist beyond 12 population doublings. 8/44 clones (18%)demonstrated continued growth extending beyond 17 doublings. All cloneswere switched to adipogenic growth conditions, and under theseconditions, 14/44 clones (32%) exhibited adipogenesis.

FIG. 6 RT-PCR analysis of gene expression in STRO-1^(bright)/VCAM-1⁺purified stromal precursor cells (MPC) isolated directly from marrowaspirates, non-induced primary stromal cultures derived from MPC(CFU-F), and CFU-F cultured under osteogenic—(BONE), chondrogenic—(CART)and adipogenic—(FAT) inductive growth conditions. Various markers of:BONE [transcription factor CBFA1; collagen type I (COLL-I);bonesialoprotein (BSP); osteopontin (OP); osteonectin (ON); osteocalcin(OCN), parathyroid hormone receptor (PTHR)]; FAT [lipoprotein lipase(LPL), transcription factor PPAR72, leptin, human adipocyte lipidbinding protein (H-ALBP)]; CARTILAGE [collagen type II (COIL-II),collagen type X (COLL-X), Aggrecam (AGGN)]. Reaction mixes weresubjected to electrophoresis on a 1.5% agarose gel and visualised byethidium bromide staining.

FIG. 7 In vitro developmental potential of MPC. Primary cultures ofderived from STRO-1^(bright)/V-1+BMMNC were cultured for 2 weeks theninduced under either osteogenic, adipocytic or chondrocytic conditionsfor 3-5 weeks. A von Kossa positive mineralised matrix formed throughoutthe cultures within 4 weeks of bone induction (200×) (A). The presenceof clusters of lipid containing adipocytes were also detected by oilred-O staining (200×) (B). Cultures were counter stained withhaematoxylin.

FIG. 8 New bone formation in vivo. ImmunoselectedSTRO-1^(bright)/VCAM-1⁺ BMMNC clones, expanded in vitro, were implantedsubcutaneously into SCID mice using porous ceramic cubes. Implants wereharvested 8 weeks post transplant. New bone formation (solid arrow) wasobserved for a proportion of clones within the cavities of the ceramiccubes (open arrow) together with surrounding fibrous and haematopoietictissue (40×) (A). The sections were counter stained with haematoxylinand eosin. A magnified view of new bone formation is shown depicting anosteocyte (arrow) (200×) (B).

FIG. 9 Dual parameter flow cytometric analysis of STRO-1⁺ human bonemarrow mononuclear cells isolated by MACS. A distinct subpopulation ofSTRO-1^(bri) cells are identified by VCAM-1, THY-1 (CD90), MUC-18(CD-146) and STRO-2.

FIG. 10. Properties of STRO-1⁺ MACS-isolated cells co-labeled withanti-CD146 (CC9). (A) Sort region, R1, represents the double positiveSTRO-1^(BRT)/CD 146⁺ population. (B) The incidence of clonogenic cellcolonies (>50 cells) based on STRO-1^(BRT)/CD146⁺ expression wasdetermined by limiting dilution analysis of 24 replicates per cellconcentration using Poisson distribution analysis from 5 independentexperiments. Forward (size) and perpendicular (granularity) lightscatter characteristics of BMMNCs (C), STRO-1^(int)/CD146⁺ cells (D) andSTRO-1^(BRT)/CD146⁺ cells (E). (F) RT-PCR analysis ofSTRO-1^(BRT)/CD146⁺ sorted marrow cells for CBFA1 (lane 2), osteocalcin(lane 4) and GAPDH (lane 6) transcripts. Control cells (BMSSC culturesgrown in the presence of dexamethasone) expressing CBFA1 (lane 1),osteocalcin (lane3), and GAPDH (lane 5) is also shown. Reaction mixeswere subjected to electrophoresis on a 1.5% agarose gel and visualisedby ethidium bromide staining. (G) In situ expression of CD 146 on bloodvessel (bv) walls (arrow) in human bone marrow (bm) sections near thebone (b) surface 20×. Sections were counter stained with Hematoxylin.(H) Dual Immunofluorescence staining demonstrating reactivity of theSTRO-1 antibody labeled with Texas red and the CC9 antibody labeled withfluorescein isothiocyanate, reacting to blood vessel walls in frozensections of human bone marrow.

FIG. 11. Immunophenotypic analysis of DPSCs in vivo. The bar graphdepicts the number of clonogenic colonies retrieved from single cellsuspensions of dental pulp following immunomagnetic bead selection basedon reactivity to antibodies that recognize STRO-1, CD146, and 3G5 andisotype-matched negative control antibodies. The data are expressed asthe number of colony-forming units obtained in the bead positive cellfractions as a percentage of the total number of colonies inunfractionated pulp cells averaged from three separate experiments.Statistical significance (*) was determined using the student t-test (p0.01) comparing the percent total number of colonies for each antibodywith the corresponding isotype-matched control.

FIG. 12. Reactivity of perivascular makers in dental pulp. (A)Immunolocalization of the STRO-1 antigen on blood vessels (small arrows)in human dental pulp (p) and around perineurium (large arrow)surrounding a nerve bundle (nb) 20×. (B) Dual Immunofluorescencestaining demonstrating reactivity of the STRO-1 antibody labeled withTexas Red to dental pulp perineurium (arrow) in combination with ananti-neurofilament antibody labeled with fluorescein isothiosyanatestaining the inner nerve bundle (nb), 40×. (C) Immunolocalization of theCD146 antigen to blood vessel walls in human dental pulp tissue 20×. (D)Dual Immunofluorescence staining demonstrating reactivity of the STRO-1antibody labeled with Texas red to a blood vessel and the CC9 antibodylabeled with fluorescein isothiosyanate. (E) Immunohistochemicalstaining of pulp tissue with a rabbit polyclonal anti-DSP antibody(arrow) to the odontoblast outer layer (od). 20×. (F) 3G5 reactivity toa single pericyte (arrow) in a blood vessel (bv) wall 40×. Tissuesections were counter stained with Hematoxylin.

FIG. 13. 3G5 reactivity to BMSSCs. (A) The representative histogramdepicts a typical dual-color FACS analysis profile of whole bone marrowmononuclear cells (BMMNCs) expressing CD 146 (PE) and 3G5 (FITC). (B)Colony efficiency assays were performed for all the different expressionpatterns observed (regions “R” 1-6). The data are expressed as the meanincidence of colony-forming units for each cell fraction averaged fromthree separate experiments.

FIG. 14. Developmental potential of purified BMSSCs and DPSCs in vivo.Cytospin preparations of MACS/FACS isolated STRO-1^(BRT)/CD146⁺ marrowcells (arrow) stained with an antibody specific to α-smooth muscle actin(A) and von Willebrand Factor (B). CD146⁺ pulp cells (large arrow)isolated by immunomagnetic bead selection (magnetic beads depicted bysmall arrows), stained with an antibody specific to α-smooth muscleactin (C) and von Willebrand Factor. (D). (E) Ectopic bone formation (b)and haematopoietic/adipogenic marrow (bm) by ex vivo expanded cellsderived from STRO-1^(BRT)/CD146⁺ BMSSCs transplanted with HA/TCP intoimmunocompromised mice for three months (E). (F) Ectopic formation ofdentin (d) and fibrous pulp tissue (p) by ex vivo expanded cells derivedfrom CD146⁺ DPSCs transplanted with HA/TCP into immunocompromised micefor three months. Sections were stained with Hematoxylin & Eosin.

FIG. 15 Expression of CD34, CD45 and Glycophorin-A on STRO-1 positivebone marrow mononuclear cells. Representative histograms depictingtypical dual-colour flow cytometric analysis profiles of STRO-1 positivebone marrow mononuclear cells isolated initially by magnetic activatedsorting and co-stained with antibodies directed against CD34 (A), CD45(B) or Glycophorin-A (C). The STRO-1 antibody was identified using agoat anti-murine IgM-fluorescein isothiocyanate while CD34, CD45 andGlycophorin-A were identified using a goat anti-murineIgG-phycoerythrin. The high expressing STRO-1 fraction which containedthe clonogenic MPC population was isolated by fluorescence activatedcell sorting based on regions R1 and R2.

FIG. 16 Bone marrow MPC are STRO-1 bright, CD34 negative, CD45 negativeand Glycophorin-A negative. The graph depicts the results of in vitroadherent colony formation assays performed for each of the differentsorted STRO-1 bright populations selected by their co-expression or lackof either the CD34, CD45 or Gycophorin-A antigens, based on regions R1and R2 as indicated in FIG. 15. These data are expressed as the meanincidence of colony-forming units for each cell fraction averaged fromtwo separate experiments.

FIG. 17 Reactivity of perivascular makers in different human tissues.Dual-colour immunofluorescence staining demonstrating reactivity of (A)STRO-1 and CD146, (B) STRO-1 and alpha-smooth muscle actin, and (C) 3G5and CD146, on blood vessels and connective tissue present on spleen,pancreas (Panel 1), brain, kidney (Panel 2), liver, heart (Panel 3) andskin (Panel 4) 20×. The STRO-1 and 3G5 antibodies were identified usinga goat anti-murine IgM-Texas Red while CD146 and alpha-smooth muscleactin were identified using a goat anti-murine or IgG-fluoresceinisothiocyanate. Co-localization is indicated by overlapping areas ofyellow and orange fluorescence (white arrows).

FIG. 18 Isolation of adipose-derived MPC by FACS. Representative flowcytometric histograms depicting the expression of STRO-1, CD146 and 3G5in fresh preparations of peripheral adipose-derived single-cellsuspensions generated following collagenase/dispase digestion aspreviously described (Shi and Gronthos 2003). The antibodies wereidentified using either a goat anti-murine IgM or IgG-phycoerythrin.Cell populations were then selected by FACS, based on their positivity(region R3) or negativity (region R2) to each marker and then platedinto regular growth medium to assess the incidence of adherentcolony-forming cells in each cell fraction.

FIG. 19 Clonogenic adipose-derived MPC are positive forSTRO-1/3G5/CD146. The bar graph depicts the number of clonogeniccolonies retrieved from single cell suspensions of enzymaticallydigested human peripheral adipose tissue, following fluorescenceactivated cell sorting, based on their reactivity to antibodies thatrecognize STRO-1, CD146, and 3G5 (FIG. 18), then cultured in standardgrowth medium as previously described for bone marrow and dental pulptissue (Shi and Gronthos 2003). The data are expressed as the number ofcolony-forming units obtained per 10³ cells plated in the positive andnegative cell fractions averaged from two separate experiments.

FIG. 20 Immunophenotypic analysis of adipose-derived MPC. Representativeflow cytometric histograms depicting the co-expression of STRO-1 andCD146 (A) and 3G5 and CD146 in fresh preparations of peripheraladipose-derived single-cell suspensions generated followingcollagenase/dispase digestion. The STRO-1 and 3G5 antibodies wereidentified using a goat anti-murine IgM-phycoerythrin while CD146 wasidentified using a goat anti-murine IgG-fluorescein isothiocyanate.Approximately 60% and 50% of the CD146 positive cells co-express STRO-1and 3G5, respectively. These data suggest that 10% or more of the CD164positive cells co-express STRO-1 and 3G5.

FIG. 21 Developmental potential of purified Adipocyte-derived MPC invitro. Preparations of primary MPC cultures derived from STRO-1⁺/CD146⁺adipose cells were re-cultured either in standard culture conditions(A), osteogenic inductive medium (B), Adipogenic inductive medium (C) orcondrogenic conditions (D) as previously described Gronthos et al. 2003.Following two weeks of multi-differentiation induction, theadipocyte-derived MPC demonstrated the capacity to form bone (B;Alizarin positive mineral deposits), fat (C; Oil Red O positive lipid)and cartilage (D: collagen type II matrix).

FIG. 22 Isolation of skin-derived MPC by FACS. Representative flowcytometric histograms depicting the expression of STRO-1, CD146 and 3G5in fresh preparations of full thickness skin-derived single-cellsuspensions generated following collagenase/dispase digestion. Theantibodies were identified using either a goat anti-murine IgM orIgG-phycoerythrin. Cell populations were then selected by FACS, based ontheir positivity (region R3) or negativity (region R2) to each markerand then plated into regular growth medium to assess the incidence ofadherent colony-forming cells in each cell fraction.

FIG. 23 Clonogenic skin-derived MPC are positive for STRO-1/3G5/CD146.The bar graph depicts the number of adherent colonies recovered fromsingle cell suspensions of enzymatically digested human skin, followingfluorescence activated cell sorting, based on their reactivity toantibodies that recognize STRO-1, CD146, and 3G5 (FIG. 15), thencultured in standard growth medium as previously described for bonemarrow and dental pulp tissue (Shi and Gronthos 2003). The data areexpressed as the number of colony-forming units obtained per 10⁵ cellsplated in the positive and negative cell fractions averaged from twoseparate experiments.

FIG. 24A. Immunophenotypic expression pattern of ex vivo expanded bonemarrow MPC. Single cell suspensions of ex vivo expanded bone marrow MPCwere prepared by trypsin/EDTA treatment then incubated with antibodiesidentifying cell lineage-associated markers. For those antibodiesidentifying intracellular antigens, cell preparations were fixed withcold 70% ethanol to permeanbilize the cellular membrane prior tostaining for intracellular markers. Isotype matched control antibodieswere treated under identical conditions. Flow cytometric analysis wasperformed using a COULTER EPICS instrument. The dot plots represent5,000 listmode events indicating the level of fluorescence intensity foreach lineage cell marker (bold line) with reference to the isotypematched negative control antibodies (thin line). B. Gene expressionprofile of cultured MPC. Single cell suspensions of ex vivo expandedbone marrow MPC were prepared by trypsin/EDTA treatment and totalcellular RNA was prepared. Using RNAzolB extraction method total RNA wasisolated and used as a template for cDNA synthesis, prepared usingstandard procedure. The expression of various transcripts was assessedby PCR amplification, using a standard protocol as described previously(Gronthos at al. 2003). Primers sets used in this study are shown inTable 2. Following amplification, each reaction mixture was analysed by1.5% agarose gel electrophoresis, and visualised by ethidium bromidestaining. Relative gene expression for each cell marker was assessedwith reference to the expression of the house-keeping gene, GAPDH, usingImageQuant software.

FIG. 25. Ex vivo expanded STRO-1^(bri) MPC can develop into arteriolesin vitro. Single cell suspensions of ex vivo expanded bone marrowSTRO-1^(bri) MPC were prepared by trypsin/EDTA treatment then platedinto 48-well plates containing 200 μl of matrigel. The STRO-1^(bri) MPCwere plated at 20,000 cells per well in serum-free medium (Gronthos etal. 2003) supplemented with the growth factors PDGF, EGF, VEGF at 10ng/ml. Following 24 hours of culture at 37° C. in 5% CO₂, the wells werewashed then fixed with 4% paraformaldehyde. Immunohistochemical studieswere subsequently performed demonstrated that the cord-like structuresexpressed alpha-smooth muscle actin identified with a goat-anti-murineIgG horse radish peroxidase antibody.

EXAMPLE 1 Isolation of Mesenchymal Precursor cells

To properly investigate the biology of BM MPC, studies were designed toisolate MPC from a heterogeneous population of unfractionated BM cells.This was achieved by using a combination of positive immunoselectionprocedures based on the unique specificity of the STRO-1 mab, in orderto maximise the recovery and purity of the MPC population. Following theisolation of homogeneous populations of MPC we then explored theirpattern of gene expression for various bone-, fat- and cartilage-relatedmarkers to determine the degree of commitment towards different stromalcell lineages in vivo. Finally we have investigated the developmentalpotential of purified populations of BM MPC in vitro under definedconditions [Gronthos et al, 1994] and in vivo by ectopic implantationinto immunodeficient mice [Haynesworth et al, 1992].

We and others have had success in isolating MPC based on theirexpression of the STRO-1 antigen either by FACS or by usingimmunomagnetic particles, such as Dynabeads [Tamayo et al, 1994] or bymagnetic-activated cell sorting (MACS) [Gronthos et al, 1995 and 1998].The latter was used initially to provide a reproducible technique forisolating BM derived MPC with the capacity to process high cell numbers.The mab STRO-1 proved to be an ideal reagent for isolating MPC fromadult BM because of its lack of reactivity to haematopoietic progenitors[Simmons and Torok-Storb, 1991 a] yielding a clean separation betweenMPC and haematopoietic progenitors in adult BM. Moreover, the antigenidentified by STRO-1 was found in the present study to be expressed atparticularly high copy number by MPC, which may in part account for thehigh efficiency and recovery of BM CFU-F observed. These studiesidentified the minor STRO-1 bright subset of the total STRO-1⁺ BMMNCfraction to contain the CFU-F population. However the resulting postMACS STRO-1^(bright) cell population was only partially enriched forMPC.

We have previously demonstrated that the cell surface antigen, VCAM-1 isuniversally expressed on BM MPC and their progeny [Simmons et al, 1992,1994]. This is in contrast to other markers expressed by BM MPC such asTHY-1, CD10, CD 13, and thrombomodulin, [Simmons et al, 1994] which arealso known to react with either haematopoietic cells and or platelets[Baum et al, 1992; Conway and Nowakowski, 1993; Ship and Look, 1993].The VCAM-1 molecule is a transmembrane glycoprotein with a molecularweight of between 95 and 110 kDa present on the membranes of stromalcells and endothelial cells [Osborn et al, 1989; Simmons et al, 1992].The immunoglobulin super family member is one ligand for the integrinreceptor α4β1 (VLA-4) present on haematopoietic stem cells, and isinvolved in the recruitment of lymphocytes and monocytes expressing α4β1to sites of infection and inflammation [Elites et al, 1990; Simmons etal, 1992]. Significantly, VCAM-1 only reacted with a minor proportion ofBMMNC effectively subletting the total STRO-1⁺ population, reactingpreferentially with the STRO-1 bright cell fraction. The BM MPCpopulation was subsequently shown to reside exclusively in theSTRO-1^(bright)/VCAM-1⁺ fraction of human adult BM.

The absolute frequency of MPC in bone marrow was determined by limitingdilution experiments using Poisson distribution statistics. Otherstudies using this statistical analysis have shown that murine BMosteoprogenitor cells with the potential to form mineralized bonenodules in vitro, occurred at a frequency of 1 per 1000 BM cells plated,based on the phenotype 5-fluoracil resistant, haematopoietic lineagemarker negative [Van Vlasselaer, 1994]. These osteoprogenitorsrepresented approximately 20% of the total MPC population in normalmurine BM [Falla et al, 1993; Van Vlasselaer, 1994]. Similar analyses offetal human BMMNC demonstrated the frequency of MPC at 1 per 1,000 to 1per 100,000 cells plated, at 14 weeks and 24 weeks gestation,respectively, based on the immunophenotype CD34⁺/CD38⁻/HLA-DR⁻ [Walleret al, 1995a]. Furthermore, additional subletting of fetal BM using thehaematopoietic marker CD50, distinguished HSC from the MPC population,but found no significant difference in the incidence of clonogenicstromal cells sorted on the basis of the phenotypeCD34⁺/CD38⁻/HLA-DR^(−/CD)50⁻ [Waller et al, 1995b]. However, no stromalprogenitors were observed when single cells of human adult BM sampleswere sorted based on the CD34⁺/CD38⁻/HLA-DR⁻ phenotype [Waller et al,1995a]. This may be due to the inefficiency of a predominantly negativeselection criteria used to isolate fetal 13M MPC and may also reflectthe use of the CD34 antigen which demonstrates low level expression onadult BM MPC [Simmons and Torok-Storb, 1991b].

In the illustrated embodiment, the incidence of clonogenic cells(clusters 10<50 cells+colonies 50) from adult human BM was determined tobe 1 per 2 STRO-1^(bright)/VCAM-1⁺ cells plated in SDM containing PDGFand EGF. Using serum-deprived medium significantly enhances theincidence of clonogenic growth over that of serum replete cultures,particularly at low plating densities [Gronthos and Simmons, 1995]. Itmust also be stated that a proportion of the wells which were scored as‘negative’ contained cell clusters of less than 10 cells. Therefore, byfurther refining the CFU-F culture assay, it may be possible tostimulate the growth of MPC in order to increase the overall purity ofthe MPC population based on the composite STRO-1^(bright)/VCAM-1⁺phenotype. Nevertheless, the combined purification technique of theillustrated embodiment effectively achieved a several thousand foldincrease in the incidence of BM MPC when compared to unfractionatedBMMNC.

The cells contained within the STRO-1^(bright)/VCAM-1⁺ BM fraction werefound to be a homogeneous population of large cells with extensivecytoplasmic processes existing in vivo in a non-cycling state. Otherstudies have found that MPC residing in the BM are almost entirelynon-cycling as shown by ³H thymidine labelling in rodents and by meansof the in vitro thymidine suicide technique in humans [Castro-Malaspinaet al, 1980; Castro-Malaspina et al, 1981]. This data coincides with theobservations that primitive multi-potential stem cells, identified inthe other cell systems such as HSC are by definition quiescent cells[Andrews et al, 1986; Szilvassy et al, 1989; Li and Johnson, 1992].Given the reported developmental potential of cultured BM MPC in vitroand in vivo the question arises as to whether these cells are trulyrepresentative of an early uncommitted phenotype with multi-potential orwhether all or a proportion of the CFU-F are already committed towards aparticular stromal cell lineage.

Analysis of the gene expression pattern of purified adult BM MPC in theillustrated embodiment has revealed that many of the genes expressed byCFU-F in vivo demonstrate a broad stromal tissue distribution related toosteoblasts, adipocytes and chondrocytes. It is very common to find inthe literature that many markers for example osteonectin, osteopontin,and alkaline phosphatase in the bone cell lineage are described as beingspecific to bone cells, when in fact these markers have a wider tissuedistribution. Therefore, it is not surprising to find that MPCidentified by STRO-1 share common markers with differentiated stromalcell types. Importantly, specific markers of commitment such as CBFA-1,collagen type II, PPARγ2, [reviewed in Roden and Harada, 1997] to bone,cartilage and fat respectively were not expressed by theSTRO-1^(bright)/VCAM-1⁺ population in fresh BM aspirates. In addition,immunohistochemical examination of STRO-1^(bright)/VCAM-1⁺ sorted BMMNCfailed to show any reactivity to the smooth muscle marker cc-smoothmuscle actin or with the endothelial marker, FVIII. Therefore the MPCresiding in the BM seem to exist in an uncommitted state, and may havethe potential under different conditions to develop into a few or all ofthe stromal elements recognised in the bone marrow microenvironment.

In the present study, cultures of purified STRO-1^(bright)/VCAM-1+ an BMCFU-F typically developed a von Kossa positive mineral by twenty onedays under osteogenic conditions (ASC-2P, PO_(4i), DEX). The presence ofmineral deposits was demonstrated in all CFU-F clones examined, where40% of the clones also displayed the capacity to differentiate intoadipocytic cell clusters. Moreover, individual CFU-F clones were alsofound to contain a small proportion of fibroblastic-like cells notassociated with either mineralization or lipid accumulation. Thesefibroblast-like cells may represent as yet undefined stromal populationssuch as reticular cells, smooth muscle cells, bone lining cells,osteocytes and committed stromal progenitors.

The developmental potential of selected CFU-F clones was furtherexamined in vivo. The porous hydroxyapatite coated ceramic cubesreproducibly supported the development of human osteogenic tissue inSCID mouse. This is in agreement with the findings in previous in vivostudies using unfractionated rodent and human BM mesenchymal cellcultures [Haynesworth et al, 1992a; Krebsbach et al, 1997; Kusnetsov etal, 1997]. In the present study, pretreating the HA ceramic cubes withpurified fibronectin was critical to maximise the number of cellsretained in the cubes after loading prior to transplantation (data notshown). Pre-treatment of HA cubes with fibronectin and laminin has beenreported to increase cell retention and spreading on the ceramic surfaceof the cubes [Dennis et al, 1992; Dennis and Caplan; 1993]. Fibronectinand laminin coated cubes were found to augment bone formation fromcultured rat BM mesenchymal cells at earlier time points in comparisonto untreated cubes [Dennis et al, 1992; Dennis and Caplan, 1993].

The present study failed to detect cartilage formation in any of thetransplantation models used, in contrast to other studies whichdemonstrated cartilage formation in diffusion chambers transplanted withrodent bone marrow or mesenchymal cells derived from the marrow of youngchildren. To date, there have been no reports describing thereproducible induction of cartilage formation using adult human bonemarrow stromal cells in vivo or in vitro. In the present study, theexpression of the hypertrophic chondrocyte marker collagen type X, bypurified adult human BM MPC, is somewhat puzzling, given the presumedspecificity of this molecule. Since the physiological role of collagentype X is unknown, its significance in bone marrow remains to bedetermined.

The present work is in accord with previous studies showing that theformation of new bone in implants of HA cubes is attributed todifferentiation of human mesenchymal cells into functional osteoblasts[Kusnetsov et al, 1997] and did not result from the recruitment ofosteoprogenitors from the surrounding host (mouse) tissue. Furthermore,other cell types present such as muscle, adipocytes and vascularendothelial cells showed no hybridization with the alu probe and aretherefore presumed to be host in origin. These findings demonstrate thata proportion of BM MPC within the STRO-1^(bright)/VCAM-1⁺ BMsubfraction, demonstrate the capacity to develop into multiple stromalcell types including osteoblasts, adipocytes and fibroblast-like cells.

Further subletting of the STRO-1^(bright)/VCAM-1⁺ BM fraction usingthree- and four-colour FACS analysis may eventually provide a means todiscriminate between subpopulations contained within the MPC pool whichexhibit different developmental potentials. The purification of MPCclones with different potential may then be used to generatemultipotent, bi-potent and uni-potent cell lines which could greatlyfacilitate the design of experimental approaches to study the molecularmechanisms regulating the commitment of early precursors into differentstromal cell lineages.

One area of potential benefit that will occur from a greaterunderstanding of the proliferation and differentiation of MPC, is theability to manipulate and expand mesenchymal cell populations in vitrofor subsequent reimplantation in vivo. The use of animal models hasdemonstrated the efficacy of utilising ex vivo expanded BM mesenchymalcells to facilitate bone regeneration and tendon repair in vivo [Bruderet al, 1998b; 1998c; Young et al, 1998]. Several studies have alsodescribed how cultured marrow stromal cells from a variety of speciesare readily infected using either amphotropic retroviruses oradenoviruses [Harigaya and Handa, 1985; Rothstein et al, 1985; Singer etal. 1987; Cicutinni et al, 1992; Roecklein and Torok-Storb, 1995]. Inaddition, some studies have demonstrated the persistence of transplantedtransduced cells over several months in animal models [Li et al, 1995;Anklesaria et al, 1996; Onyia et al, 1998 Reiw et al, 1998]. Thereforethe ability to harvest purified human MPC from aspirates of BM and toexpand these cells ex vivo makes them ideal candidates as possiblevehicles for gene transfer, in order to treat a variety of diseases andgenetic disorders.

Materials and Methods

Subjects

Aspirates of human BM samples were obtained from the iliac crest and thesternum of normal adult volunteers with their informed consent,according to procedures approved by the ethics committee at the RoyalAdelaide Hospital, South Australia. Bone marrow mononuclear cells(BMMNC) were obtained by centrifugation over Ficoll 1.077 g/ml(Lymphoprep, Nycomed, Oslo, Norway) at 400 g for 30 minutes (min) andthen washed and resuspended with Hank's buffered saline solutioncontaining 1% bovine serum albumin and 10 mM HEPES, pH 7.35 (HBSS).

Isolation of STRO-1⁺ Cells by Magnetic-Activated Cell Sorting (MACS)

This procedure is a modification of that described elsewhere [Gronthoset al, 1998). Approximately 1×10⁸ BMMNC were incubated with STRO-1supernatant for 60 min on ice. The cells were then washed in HBSS andresuspended in 1 ml of HBSS containing a 1/50 dilution of biotinylatedgoat anti-mouse IgM (μ-chain specific; Southern BiotechnologyAssociates, Birmingham, Ala.) for 45 min on ice. Following this, thecells were washed twice in MACS buffer (single strength Ca²⁺ and Mn²⁺free PBS supplemented with 1% BSA, 5 mM EDTA and 0.01% sodium azide) andresuspended in 900 μl of MACS buffer to which 100 μl of streptavidinmicrobeads (Miltenyi Biotec, Bergiach Gladbach, F.R.G.) was added. Thecells were further incubated for 15 min on ice after whichstreptavidin-fluorescein isothiosyanate (FITC) conjugate (1/50; CaltagLaboratories, San Francisco, Calif.) was added directly to thesuspension for an additional 5 min. The cells were separated on a MiniMACS magnetic column (column capacity 10⁷ cells, Miltenyi Biotec)according to the manufacturers specifications.

Purification of the CFU-F Population by Fluorescence Activated CellSorting (FACS)

Dual colour-FACS analysis of the STRO-1^(bright) population was achievedby incubating the MACS isolated STRO-1 population with saturating levelsof the Mab 6G10 (mouse IgG1 anti-human CD106: vascular endothelialadhesion molecule-1, VCAM-1; kindly donated by Dr. B. Masinovski FCOSCorp., Seattle, Wash.) for 30 min on ice. After washing with HBSS thecells were incubated with a second label goat anti-mouse IgG (γ-chainspecific) phycoerythrin (PE) conjugate antibody (1/50; SouthernBiotechnology Associates, Birmingham, Ala.) and a streptavidin-FITCconjugate (1/50; CALTAG Laboratories, San Francisco, Calif.) for 20 minon ice. The cells were then washed in HBSS prior to being sorted usingthe automated cell deposition unit (ACDU) of a FACStar^(PLUS) (BectonDickinson, Sunnyvale, Calif.) flow cytometer. STRO-1^(bright)/VCAM-1⁺cells were seeded at plating densities of 1, 2, 3, 4, 5, and 10 cellsper well (96-well plates) in replicates of 24 wells per plating density(FIG. 2). The cells were cultured in serum deprived medium onfibronectin coated wells as previously described [Gronthos and Simmons1995; Gronthos et al, 1998]. On day 10 of culture the cells were thenfixed and stained for 60 min with 0.1% toluidine blue in 1%paraformaldehyde. Aggregates of 50 cells were scored as CFU-F coloniesand aggregates of 10<50 cells were scored as clusters using an OlympusSZ-PT dissecting light microscope (Olympus Optical Co. Ltd, Tokyo,Japan).

Analysis of Cell Cycling Status of STRO-1+ BMMNC

The STRO-1⁺ BMMNC were isolated by MACS as described above and thenincubated with streptavidin PE for 15 min on ice. After washing twicewith PBS the cells were fixed for 10 min with cold methanol (70%) onice. Following this, the cells were washed three times with PBS and thenincubated in blocking buffer for 15 minutes. The monoclonal antibodyKi-67 conjugated to FITC (DAKOPATTS A/S, Glostrup, Denmark) was addeddirectly to the cells (1/10 dilution) in blocking buffer for 45 min onice served as the negative control.

RNA Isolation and First-Strand cDNA Synthesis

Total cellular RNA was routinely prepared from 2×10⁴STRO-1^(bright)/VCAM-1⁺ cells collected as a bulk population and lysedusing RNAzolB extraction method (Biotecx Lab. Inc., Houston, Tx.), asper manufacturers recommendations. RNA isolated from each subpopulationwas then used as a template for cDNA synthesis. cDNA was prepared usinga First-strand cDNA synthesis kit from Pharmacia Biotech (Uppsala,Sweden) according to manufacturers instructions. Briefly, total RNA wasresuspended in 8 μl of DEPC-treated water and subsequently heated to 65°C. for 10 min. Following snap cooling on ice, the RNA was added to 7 μlof premix containing reaction buffer, oligo-dT as primer and SuperscriptMMLV Reverse transcriptase. Following incubation at 42° C. for 60 min,the volume of the reaction was adjusted to 50 μl with the addition of 35μl of sterile water. The samples were stored at −20° C.

Polymerase Chain Reaction (PCR)

Due to limiting cell numbers, the expression of various bone-relatedtranscripts (Table 1) was assessed by polymerase chain reaction (PCR)amplification, using a standard protocol [Sambrook et al, 1989]. Twomicrolitres of first strand cDNA mixture from each subpopulation wasdiluted in a 50 μl PCR reaction (67 mM Tris HCl pH 8.8, 16.6 mM(NH₄)₂SO₄, 0.45% Triton X100, 200 μg/ml gelatin, 2 mM MgCl₂, 200 μM eachdNTP) containing 100 ng of each primer (Table 1), to which 2.5 units ofAmplitaq DNA Polymerase (Perkin-Elmer, Norwalk, Conn., USA) was added.Reaction mixes were overlayed with mineral oil and amplificationachieved by incubation in a Perkin-Elmer/Cetus thermal cycler. Primerdesign enabled typical cycling conditions of 94° C./(2 min), 60° C./(30sec), 72° C./(1 min) for 40 cycles, with a final 10 min incubation at72° C. To control for the integrity of the various RNA preparations, theexpression of GAPDH and/or beta-2-microglobulin was also assessed.Following amplification, 10 μl of each reaction mixture was analysed by1.5% agarose gel electrophoresis, and visualised by ethidium bromidestaining.

The Developmental Potential of BM CFU-F in Vitro

We have previously reported the conditions for the induction of humanbone marrow stromal cells to develop a mineralised bone matrix in vitro[Gronthos et al, 1994]. Briefly, the osteogenic and adipocytic potentialof thirty day 4 CFU-F clones derived from single STRO-1^(bright)/VCAM-1⁺sorted cells was assessed by culturing in alpha modification of Eagle'smedium (α-MEM: Flow Laboratories) supplemented with 20% FCS, L-glutamine(2 mM), β-mercaptoethanol (5×10⁻⁵ M), L-ascorbic acid 2-phosphate (100μM) (ASC-2P: Novachem, Melbourne, Australia), dexamethasone sodiumphosphate (10⁻⁸M) (DEX: David Bull Laboratories; Sydney, Australia),KH₂PO₄ (1.8 mM) (BDH Chemicals) and Hepes (10 mM), at 37° C., 5% CO₂.The media was changed twice a week for a period of six weeks. Cultureswere rinsed twice with PBS then fixed in situ with 10% neutral formalinfor 30 mon. Staining for vonKossa was performed according to the methodof Pearse and Gardner (1972). Sections or culture wells were washedtwice in distilled water and then stained in 5% aqueous AgNO₃ for 60 minunder ultraviolet light. After staining with AgNO₃, the sections werewashed twice with distilled water and then placed in 5% sodiumthiosulphate for 1 min. Cultures were washed in distilled water, counterstained with Mayer's haematoxylin and mounted. Oil Red O (ORO) stainingwas performed as described by Grimble (1998). Briefly, cultures werefixed as described above, washed twice with PBS and air dried. Cultureswere immersed in a solution 0.5% (w/w) ORO in isopropanol for 15 min atroom temp., washed three times with distilled water and subsequentlycounterstained with haematoxylin.

Similarly, the chondrogenic potential of the same clones was assessed byculturing 2×10⁵ cells per clone in 0.5 mls SDM supplemented with TGFβ1and gently centrifuged at 200 g for 2 min in a 10 ml polypropolene tubethen incubated at 37° C., 5% CO₂. The media was changed twice a week fora period of three weeks

The Developmental Potential of BM CFU-F in Vivo

Bulk cultures of CFU-F derived from STRO-1^(bright)/VCAM-1⁺ sorted BMMNCwere cultured for 5 weeks in the presence of ASC-2P and DEX and 10% FCS.The adherent cell layers were trypsinised and seeded onto 27mm³ poroushydroxyapatite ceramic cubes (Zimmer Corporation, Warsaw, Ind., USA)pre-coated with fibronectin (5 μg/ml) (Boehringer Mannheim, Germany).The ceramic cubes were then implanted into subcutaneous pockets into thebacks of SCID mice for a period of up to 8 weeks as described previously[Haynesworth et al, 1994; Kuznetsov et al, 1997]. Recovered implantswere fixed in 10% buffered formalin for 2 days then decalcified for afurther seven days in 0.5M EDTA before being embedded in paraffin wax.Cross-sections of the cubes were prepared as 5 μm sections onto glassslides pre-coated with Cell-Tak and counter stained with haematoxylinand eosin.

In Situ Hybridization for the Human Specific Alu Sequence

The HA ceramic implants were recovered 8 weeks post transplant andprepared for paraffin embedding on Cell-Tak coated slides as describedabove. To determine the origin of the cells within the implants in situhybridization analysis was performed using a DNA probe specific to theunique human repetitive alu sequence (Kuznetsov et al, 1997]. The humanspecific alu sequence (pBLUR8; ATCC) was subcloned into the BamH1restriction site of a pGEM-4Z plasmid (Promega). Thedigoxigennin-labeled alu specific probe was prepared by PCR containing1× PCR buffer (67 mM Tris HCl pH 8.8, 16.6 mM (NH₄)₂SO₄, 0.5% Triton-X100, 0.2 μg/ml gelatin, 2.5 mM MgCl₂, 0.2 mM dATP, 0.2 mM dCTP, 0.2 mMdGTP, 1.9 mM dTTP, 0.1 mM digoxygenin-11-dUTP (Boehringer Mannheim), and0.25 units of Amplitaq DNA Polymerase) and 100 ng of SP6 and T7 primers(Table 1) and 1 ng of plasmid DNA (pGEM-4Z; Promega Corp., Madison,Wis.) containing the alu sequence subcloned into the BamHI restrictionsite from (pBLUR8; ATCC, Rockville, Md.). Sections were deparaffinizedwith xylene and ethanol then rehydrated through graded (100%, 90%, 70%,50%) ethanol solutions. The sections were then treated with 0.2N for 7min at room temperature and then incubated in 1 mg/ml pepsin (Sigma, St.Louis, Mo.) in 0.1N HCl for 10 minutes at 37° C. After washing in PBS,the sections were treated with 0.25% acetic acid containing 0.1Mtriethanolamine (pH 8.0) for 10 min and prehybridized with 50% deionizedformamide containing 4× SSC for 15 min at 37° C. The hybridizationsolution (I ng/μl digoxigenin-labeled probe in 1× Denhardt's solution,5% dextrane sulfate, 0.2mg/ml, salmon sperm DNA, 4× SSC, 50-% deionizedformamide) was then added to the sections for denaturation at 95° C. for3 minutes followed by hybridization at 45° C. for 3 hr. After washingwith 2× SSC and 0.1× SSC, digoxigenin-labeled DNA was detected byimmunohistochemistry using antidigoxigenin alkalinephosphatase-conjugated Fab fragments (1/5000; Boehringer Mannheim Corp.,GMBH, Germany) followed by incubation with the corresponding alkalinephosphatase nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphatesubstrate solution as recommended by Boehringer Mannheim. Micrographswere taken with Ektachrome 64 T colour film using an Olympus IMT-2inverted light microscope.

Telomerase Repeat Amplification Protocol (TRAP) Assay

Telomerase activity was measured by a modified non-radioactive TRAPprotocol essentially as described by Fong et al (1997). Telomerase cellextracts were prepared by the method of Kim et al, (1994), with minormodifications. Populations of sorted or cultured cells were lysed inice-cold CHAPS extraction buffer (0.5%3[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate], 50 mMTris-HCl, pH 7.4, 5 mM MgCl₂, 5 mM EGTA, 25 mM 2-mercaptoethanol, 1ng/ml leupeptin, and 50% glycerol in DEPC-treated water), at aconcentration of 1000 cells/μl, incubated on ice for 30 minutes andcentrifuged at 16000×g for 20 minutes at 4° C., the supernatantrecovered and stored at −80° C. until required. Detection of telomeraseactivity was performed in a two-step process as previously described(Fong et al, 1997). Briefly, to 2 μl of cell extract, 16.5 μl of TRAPreaction buffer (20 mM Tris-HCl, pH8.2, 1.5 mM MgCl₂, 63 mM KCl,0.05%Tween-20, 1 mM EGTA), 100 ng of each of TS primer(5′-AATCCGTCGAGCAGAGTT-3′), and CX-ext primer(5′-GTGCCCTTCCCTTACCCTTACCC TAA-3′), 0.5 μL dNTPs (10 mM stock) wereadded, and the reaction mix incubated at 25° C. for 30 minutes.Telomerase was subsequently inactivated by heating the reaction to 90°C. for 2 minutes, prior to the addition of 5 μl of PCR mixture,containing 3.5 μl of TRAP reaction buffer, 1 μl of CX-ext primer and 2.5U Taq polymerase. Reaction mixes were covered with mineral oil andplaced in a Hybaid thermocycler, and subjected for 34 cycles of 94° C.for 30 seconds, 50° C. for 30 seconds and 72° C. for 45 seconds, with afinal extension at 72° C. for 2 minutes. To confirm the specificity ofthe telomerase products, in all cases, a 2 μl aliquot of each CHAPSlysate was subjected to denaturation by heating samples at 100° C. for10 minutes. 25 μl of each reaction was resolved on a non-denaturing 12%polyacryalmide gel, and visualised by staining width SYBR Greenfluorescent dye (FMC Bioproducts, Oreg., USA) as recommended by themanufacturer. The TRAP products were analysed using a fluorescencescanning system (Molecular Dynamics, Sunnyvale, Calif., USA).

Transmission Electron Microscopy (TEM) STRO-1^(bright)/VCAM-1⁺ cells(approximately 2×10⁴ cells) were collected as a bulk population intoeppendorf microtubes, washed once in 0.05M sodium cacodylate buffer andthen fixed in 2.5% glutaraldehyde (EM Grade) in cacodylate buffer for 2hr. The cultures were postfixed with 2% osmium tetroxide (VIII) (BDHChemicals) in cacodylate buffer for 1 hr. After this, the cultures weredehydrated with graded ethanol solutions (70%, 90%, 100%). Epoxy resin(TAAB Laboratories; Berkshire, England) was then used to infiltrate thecultures overnight at 37° C. Polymerization of the resin was carried outat 60° C. for 24 hr under vacuum. Ultrathin sections were cut on a LKB8800 Ultrotome II (Broma, UK) and mounted onto copper grids. Sectionswere then examined using a JEOL 1200 EX II (Tokyo, Japan) transmissionelectron microscope. Photographs were taken using ILFORD EM Technicalfilm.

Results

Isolation and Purification of STRO-1⁺ BM MPC

We have previously demonstrated the effectiveness of MACS to isolate andenrich for MPC from aspirates of human BM based on the cell surfaceexpression of the STRO-1 antibody [Gronthos and Simmons, 1995; Gronthoset al, 1998]. In the present study, flow cytometric analysis of MACSisolated STRO-1⁺ BMMNC cells demonstrated a heterogeneous pattern ofexpression spanning over four logs in fluorescence intensity (FIG. 1).Single-color FACS was subsequently employed to sort the STRO-1⁺ BMMNCfraction into three subsets; STRO-1^(dull) STRO-1^(intermediate) andSTRO-1^(bright). Clonogenic assay for CFU-F in the different sortedSTRO-1⁺ subpopulations demonstrated that the majority of the MPC werecontained within the STRO-1^(bright) cell fraction. There was a 900 foldincrease in the incidence of CFU-F in the STRO-1^(bright) populationwhen compared to unfractionated BMMNC (Table 1) demonstrating that BMMPC contained a high copy number of the STRO-1 antigen on their cellsurface. The recovery of the MPC population in the STRO-1^(bright)fraction was >75% in respect to the estimated total number of CFU-F inthe BM sample pre-MACS.

We attempted to obtain a more accurate discrimination of theSTRO-1^(bright) subset by incubating the total STRO-1⁺ MACS isolatedcells with the stromal cell surface antigen VCAM-1 (FIG. 2A) previouslyfound to react exclusively with BM MPC [Simmons et al, 1994]. Dualcolor-FACS was used to identify and isolate the STRO-1^(bright)/VCAM-1⁺BMMNC fraction. Limiting dilution analysis was subsequently performed,using the FACStar^(PLUS) automated cell deposition unit, to seedSTRO-1^(bright)/VCAM-1⁺ cells at various plating densities as describedin the methods. Cells were cultured under serum deprived conditions inthe presence of PDGF and EGF (10 ng/ml) previously found to support theclonogenic growth of CFU-F above that of serum replete conditionsparticularly at low plating densities [Gronthos and Simmons, 1995]. Themean incidence (n=6 different BM donors) of day 10 CFU-F colonies (>50cells) was determined to be 1 CFU-F per 3 STRO1^(bright)/VCAM-1⁺ cellsplated using Poisson distribution statistics (FIG. 2B). Furthermore, theincidence of clonogenic cells (clusters>10<50cells+colonies) was foundto be 1 per 2 STRO-1^(bright)/VCAM-1⁺ cells plated (FIG. 2C). TheMACS/FACS purification technique effectively achieved a 5×10³ foldenrichment of the CFU-F population when compared to unfractionated BMMNCwith an average incidence of 1 CFU-F colony per 10⁴ BMMNC. It must alsobe stated that a proportion of the wells which were scored as ‘negative’contained cell clusters of less than 10 cells.

Characterization of Purified BM MPC

Morphological examination of freshly sorted STRO-1^(bright)/VCAM-1⁺cells was carried out by transmission electron microscopy. Purified BMCFU-F appeared to be a homogeneous population of large cells containingmany cytoplasmic processes and a large nucleous with an open chromatinstructure (FIG. 3). To determine the cell cycling status of the CFU-Fpopulation in aspirates of BM the MACS isolated STRO-1⁺ BMMNC fractionwas further incubated with the cell cycling specific antigen Ki-67[Gerdes et al, 1984; Wersto et al, 1988]. Two color flow cytometricanalysis revealed that the STRO-1^(bright) subset which contained theCFU-F population lacked co-expression of the Ki-67 antigen demonstratingthat these cells are non-dividing in vivo (FIG. 4A). Telomerase activitywas examined in cell extracts from sorted and cultured candidate stromalprogenitor cell populations by a modified TRAP assay. Telomeraseactivity was present in all fractions including the candidate stromalstem cell compartment isolated from adult bone marrow, defined by theirexpression of both the STRO-1 and VCAM-1 (CD106) cell surface molecules(FIG. 4B).

To assess the proliferative capacity of BM MPC, individual CFU-Fcolonies (n=44) derived from two BM samples were expanded in thepresence of serum under normal clonogenic growth conditions. A minorproportion of clones (8/44, 18%) demonstrated continued growth extendingbeyond 20 population doublings while the remainder showed little or noproliferation beyond 12 population doublings (FIG. 5). These cells alsoappeared to be capable of differentiating into adipose cells, whereasother isolated cells were less likely to do so.

A detailed phenotypic analysis of freshly isolated BM MPC pre-culturewas compiled. Total RNA obtained from STRO-1^(bright)/VCAM-1⁺ cells wasused to generate full-length first-strand cDNA as described in themethods. RT-PCR analysis revealed the presence of various bone cellmarkers including bonesialoprotein, osteonectin, and collagen type I.However, there was an absence in the expression of osteopontin, theparathyroid hormone receptor, and the more specific bone cell markersosteocalcin and the transcription factor CBFAI (FIG. 6A). Similarly, thefat-related markers lipoprotein lipase and the adipocyte human lipidbinding protein were found to be expressed by theSTRO-1^(bright)/VCAM-1⁺ population but there was no detectableexpression of the adipocyte specific markers, the obese gene product(leptin) and the early transcription factor PPARγ2 in these cells (FIG.6B). Furthermore the cartilage specific markers collagen type U andaggrecan were also not expressed by our purified MPC population. Howeverthe STRO-1^(bright)/VCAM-1⁺ cell fraction was found to express collagentype X, a marker associated with hypertrophic chondrocytes (FIG. 6C). Inaddition, cytospin preparations of STRO-1^(bright)/VCAM-1⁺ sorted BMMNCfailed to show any reactivity to the smooth muscle marker α-smoothmuscle actin or with the endothelial marker, FVIU (data not shown).Overall the MPC population appeared to represent an early precursorpopulation not yet fully committed to anyone particular stromal celllineage.

Culture expanded bulk CFU-F derived from STRO-1^(bright)/VCAM-1⁺ sortedcells were assessed for their ability to develop into functionalosteoblasts, chondrocytes and adipocytes in vitro as previouslydescribed [Gronthos et al, 1994]. A von Kossa positive mineralisedmatrix developed throughout the cultures by the end of the sixth week ofinduction (FIG. 7A). In addition, clusters of Oil Red O positiveadipocytes were observed within the adherent layers in the same cultures(FIG. 7B). Following three weeks of chondrocytic induction in thepresence of TGFβ1, the cells were also found to express the cartilagespecific marker collagen type II by immunohistochemistry. FurthermoreRT-PCR analysis of total RNA isolated from the different cultureconditions demonstrated the expression of markers specific to bone(CBFA-1, OCN, PTH-R), fat (PPARγ2, leptin) and cartilage (collagen typeII, aggrecan) (FIG. 6B).

The Developmental Potential of BM MPC Clones in Vitro and in Vivo

Bone marrow CFU-F clones were established from STRO-1^(bright)/VCAM-1⁺sorted cells from three individual BM donors. At day 4 of culture,single clonogenic clusters were identified and expanded by subculture.Half of the cells from the first passage were taken from each clone andcultured under osteogenic growth conditions as described above. Theosteogenic potential of ninety CFU-F clones was assessed where a vonKossa positive mineralised matrix formed in all of the ninety clones.However, only a proportion (38%±15SEM, n=3) of the same clones gave riseto clusters of lipid containing oil red-O positive adipocytesdemonstrating the bi-potential of the CFU-F population in vitro.

Half the cells from a representative 46 clones were subcultured andexpanded for several weeks, then seeded into porous HA ceramic cubes andimplanted subcutaneously into SCID mice for a period of 8 weeks aspreviously described [Haynesworth et al, 1992, Kusnetsov et al, 1997].Cross-sections of the cubes prepared for histiological examinationshowed that all of the implants contained an extensive network of bloodvessels and fibrous tissue (FIG. 8A and FIG. 8B). Bone formation wasfound in 42% (n=26) and 55% (n=20) of the clones isolated from twodifferent BM aspirates. The ability of individual MPC clones to form avon Kossa positive mineralised matrix in vitro did not always correlateto the development of new bone in vivo. Similarly, the capacity of MPCclones to form adipocytic clusters in vitro had no bearing on thedevelopment of new bone formation in vivo.

The origin of the cellular material within the recovered implants wasassessed by in situ hybridization using a DNA probe specific to theunique human repetitive alu sequence. The fibrous tissue, bone liningcells and osteocytes within the newly formed bone were all found to bepositive for the alu sequence confirming their human origin and thebi-potential of a proportion of BM MPC (FIG. 9C and FIG. 9D).Conversely, the fat and smooth muscle surrounding the ceramic cubes didnot express the alu sequence and was therefore presumed to haveoriginated from the host. Similarly, the endothelium lining the smallblood vessels were also negative for the alu sequence implying they werederived from the mouse vasculature. In addition, there was no cartilageformation observed in sections of different implants and at differenttime points, as assessed by immunohistochemical analysis using apolyclonal antibody specific for collagen type II (data not shown).

Uses of MPCs

EXAMPLE 2 Repair of Articular Cartilage

Damaged articular cartilage generated by trauma or by diseases such asosteoarthritis and rheumatoid arthritis usually does not heal. Howeverit is expected that this type of defect could be treated by implantingcultured MPCs of the present invention into the defect. The carrier maybe pliable to mould to the shape of the defect and to promote round cellshape which is important for induction of chondrocyte differentiation. Asuitable carrier may be constructed of collagen or fibrin. See Caplan etal. in U.S. Pat. No. 5,226,914.

EXAMPLE 3 Repair of Bone

A combination of MPCs as well as a suitable support can be introducedinto a site requiring bone formation. Cultured MPCs contained in calciumphosphate ceramic vehicles may be implanted into the defect site. Forappropriate methods and techniques see Caplan et al in U.S. Pat. No.5,226,914 and U.S. Pat. No. 5,837,539.

EXAMPLE 4 Anchoring of Prosthetic Devices

The surface of a prosthetic device can be coated with MPCs prior toimplantation. The MSCs can then differentiate into osteogenic cells tothereby speed up the process of bony ingrowth and incorporation of theprosthetic device. See Caplan et al. in U.S. Pat. No. 5,226,914 and U.S.Pat. No. 5,837,539.

EXAMPLE 5 Gene Therapy

An exogenous nucleic acid that encodes a protein or peptide withtherapeutic may be transformed into the enriched population usingstandard techniques (see U.S. Pat. No. 5,591,625 by Gerson et al.). Thetransformed cell population can then be introduced into the body of thepatient to treat a disease or condition. For example, can be used toprovide a continuous delivery of insulin, or genes encoding Factor VIIIwhich is involved in clotting and therefore may be used inhaemophiliacs.

EXAMPLE 6 Marrow Transplantation

A composition containing purified MPCs can be injected into a patientundergoing marrow transplantation prior to the introduction of the wholemarrow. In this way the rate of haemopoiesis may be increased,particularly following radiation or chemotherapy. The composition mightalso include haematopoietic cells for use in radiotherapy orchemotherapy.

EXAMPLE 7 Isolation and Expansion of Precursor Cells

Stem cell niches identified in a number of different adult tissuesincluding skin, hair follicles, bone marrow, intestine, brain, pancreasand more recently dental pulp, are often highly vascularized sites.⁽¹⁾The maintenance and regulation of normally quiescent stem cellpopulations is tightly controlled by the local microenvironmentaccording to the requirements of the host tissue.^((2,3)) Both thesupportive connective tissues of bone marrow and dental pulp containstromal stem cell populations with high proliferative potentials capableof regenerating their respective microenvironments with remarkablefidelity, including the surrounding mineralized structures of bone anddentin.^((4,5)) In the postnatal organism, bone marrow stroma exists asa loosely woven, highly vascularized tissue that supports and regulateshematopoiesis.⁽⁶⁻⁸⁾ At a time when many tissues have lost or decreasedtheir ability to regenerate, adult bone marrow retains a capacity forcontinuous renewal of haematopoietic parenchymal tissue and isresponsible for remodeling the adjoining bone surfaces.^(9,10)) Incontrast, the inner pulp chamber of teeth is comprised of anon-haematopoietic, compact fibrous tissue, infiltrated by amicrovascular network, that is entombed by mineralized dentin.⁽¹¹⁻¹³⁾Following tooth maturation, dental pulp becomes relatively static,acting only in a reparative capacity in response to a compromised dentinmatrix caused by insults such as caries or mechanical trauma.

Precursors of functional osteoblasts (BMSSCs: bone marrow stromal stemcells) and odontoblasts (DPSCs: dental pulp stem cells), both forms ofMPCs identified by their source tissue, were initially identified bytheir capacity to form clonogenic cell clusters in vitro, a commonfeature amongst different stem cell populations.^((4,14-18)) The progenyof ex vivo expanded BMSSCs and DPSCs share a similar gene expressionprofile for a variety of transcriptional regulators, extracellularmatrix proteins, growth factors/receptors, cell adhesion molecules, andsome, but not all lineage markers characteristic of fibroblasts,endothelial cells, smooth muscle cells and osteoblasts.^((4,19))However, previous studies have documented that individual BMSSC coloniesdemonstrate marked differences in their proliferation rates in vitro anddevelopmental potentials in vivo.^((5,14,20)) Similar to these findings,we have recently observed comparable levels of heterogeneity in thegrowth and developmental capacity of different DPSC colonies.⁽²¹⁾Together, these studies infer a hierarchical arrangement of stromalprecursor cells residing in bone marrow and dental pulp, headed by aminor population of highly proliferative pluri-potential stem cells thatgive rise to committed bi- and uni-potential progenitor cellpopulations.⁽²²⁾

Despite our extensive knowledge about the properties of cultured BMSSCsand DPSCs, we still do not know if their in vitro characteristics are anaccurate portrait of their true gene expression patterns anddevelopmental potentials in situ. In addition, it is not formally knownif all of the colony-forming cells within each tissue are derived fromone pluri-potent stem cell pool or whether they arise from committedprogenitors belonging to distinct lineages. There is also a lack ofinformation regarding the precise anatomical location of BMSSCs andDPSCs in their respective tissues. This is mainly attributed to therarity of stem cells and the absence of specific markers that identifydifferent developmental stages during osteogenesis and odontogenesis,particularly for primitive subpopulations. It has previously beenhypothesized that one possible niche for precursors of osteoblasts andodontoblasts may be the microvasculature networks of bone marrow anddental pulp, respectively.^((23,24))

Materials and Methods

Tissue Samples

Iliac crest-derived bone marrow mononuclear cells (BMMNCs), from normalhuman adult volunteers were obtained under guidelines set by the RoyalAdealaide Hospital Human Ethics Committee. Normal human impacted thirdmolars were collected from young adults the University of AdelaideDental Clinic Research under approved guidelines set by the Universityof Adelaide Human Ethics Committee, respectively. Discarded fullthickness skin and peripheral adipose tissue were obtained from routineplastic surgery procedures from the Skin Cell Engineering Laboratory,under the guidelines set by the Royal Adelaide Hospital Human EthicsCommittee. The pulp tissue was separated from the crown and root aspreviously described.⁽⁴⁾ Single cell suspensions of dental pulp, skinand adipose tissue were prepared by enzymatic digestion in a solution of3 mg/ml collagenase type I (Worthington Biochem, Freehold, N.J.) and 4mg/ml dispase (Boehringer Mannheim, GMBH, Germany) for one to threehours at 37° C. Single cell suspensions were obtained by passing thecells through a 70 μm strainer (Falcon, BD Labware, Franklin Lakes,N.J.). Cell (0.01 to 1×10³/well) preparations of bone marrow, dentalpulp, skin and adipose were then used for either, immunoselection, RNAextraction, or direct culture in 6-well plates (Costar, Cambridge,Mass.) as described below.

Other human tissue specimens (Brain, liver, heart, kidney, lung, spleen,thymus, lymph node, pancreas, skin) were obtained from autopsies carriedout at the Royal Adelaide Hospital during routine pathologicalexaminations under approved guidelines set by the Royal AdelaideHospital Human Ethics Committee. Small specimens approximately 0.5 cm²of each tissue type were placed into Tissue-Tek II cryomoulds 25 mm×20mm×5 mm (Miles Laboratories; Naperville, Ill.) and embedded with O.C.T.compound medium (Miles Laboratories) by immersion into a 150 ml to 200ml pyrex glass beaker of iso-pentane (BDH Chemicals, Poole, England)pre-cooled by suspending a glass beaker into a bath of liquid nitrogen.The isopentane has cooled when the bottom of the glass is white. Thefrozen sections were immediately stored at −80° C. Frozen sections ofnerve and muscle tissue were obtained from the Histopathology Departmentof the I.M.V.S., South Australia and sections of foreskin were obtainedfrom the Immunology Department of the I.M.V.S., South Australia.Sections of formalin fixed, paraffin embedded human foetal limb (52days) were kindly provided by Dr. T. J. Khong from the Department ofHistopathology, Women's and Children's Hospital, Adelaide, SouthAustralia.

Colony Efficiency Assay and Culture

Single cell suspensions were plated at low plating densities (between1,000 and 10,000 cells per well, as triplicates in six well plates) toassess colony-forming efficiency of different immunoselected cellfractions. The cells were cultured in alpha-Modification of Eagle'sMedium supplemented with 20% foetal calf serum, 2 mM L-Glutamine, 100 μML-ascorbate-2-phosphate, 100 U/ml penicillin and 100 mg/ml streptomycinat 37° C. in 5% CO₂. Day 14 cultures were fixed with 4% formalin, andthen stained with 0.1% toluidine blue. Aggregates of equal to or greaterthan fifty cells were scored as clonogenic colonies equivalent to colonyforming units-fibroblastic (CFU-F).

Magnetic-Activated Cell Sorting (MACS)

This procedure is a modification of that described elsewhere.⁽²⁵⁾Briefly, approximately 1×10⁸ BMMNCs were incubated with STRO-1brisupernatant (murine anti-human BMSSCs, IgM)⁽²⁹⁾ (1/2) for 1 hour on ice.The cells were then washed with PBS/5% FBS and resuspended in a 1/50dilution of biotinylated goat anti-mouse IgM (μ-chain specific; CaltagLaboratories, Burlingame, Calif.) for 45 minutes on ice. After washing,the cells were incubated with streptavidin microbeads (Miltenyi Biotec,Bergisch Gladbach, F.R.G.) for 15 minutes on ice, then separated an aMini MACS magnetic column (Miltenyi Biotec) according to themanufacturers recommendations.

Fluorescence Activated Cell Sorting (FACS) STRO-1bri MACS isolated cellswere incubated with a streptavidin-FITC conjugate (1/50; CALTAGLaboratories) for 20 minutes on ice then washed with PBS/5% FBS.Single-color fluorescence activated cell sorting (FACS) was performedusing a FACStar^(PLUS) flow cytometer (Becton Dickinson, Sunnyvale,Calif). Dual color-FACS analysis was achieved by incubatingMACS-isolated STRO-1^(bri) BMMNCs with saturating (1:1) levels of CC9antibody supernatant (mouse anti-human CD146/MUC-18/Mel-CAM, Dr. StanGronthos) for one hour on ice. After washing with PBS/5% FBS, the cellswere incubated with a second label goat anti-mouse IgG₂a (γ-chainspecific) phycoerythrin (PE) conjugate antibody (1/50, CALTAGLaboratories) for 20 minutes on ice. The cells were then sorted usingthe automated cell deposition unit (ACDU) of a FACStar^(PLUS) flowcytometer. Limiting dilution assay: seeded 1, 2, 3 4, 5, & 10 cells perwell, 24 replicates, cultured in serum-deprived medium for 10 days aspreviously described⁽²⁶⁾. Similarly, freshly prepared unfractionatedBMMNCs were incubated with CC9 (IgG_(2a)) and 3G5 (IgM) antibodies orisotype-matched negative control antibodies for one hour on ice. Afterwashing with PBS/5% FBS, the cells were incubated with a second labelgoat anti-mouse IgG_(2a) (γ-chain specific) phycoerythrin (PE) and IgM(1/50; CALTAG Laboratories) conjugated antibodies for 30 minutes on ice.Cells were washed in PBS/%5 FBS prior to being analysed using aFACStar^(PLUS) flow cytometer. Positive reactivity for each antibody wasdefined as the level of fluorescence greater than 99% of the isotypematched control antibodies.

Flow Cytometric Analysis

Single cell suspensions of ex vivo expanded bone marrow MPC wereprepared by trypsin/EDTA treatment then incubated with neat STRO-1supernatant or antibodies identifying different cell lineage-associatedmarkers (10 mg/ml) for one hour on ice. The cells were then washed inPBS/5% FBS then incubated either with a goat anti-murineIgM-phycoerythrin (1/50, SouthernBiotechnologies), goat anti-murine oranti-rabbit IgG-phycoerythrin (Caltag Laboratories). For thoseantibodies identifying intracellular antigens, cell preparations werepermeanbilize the cellular membrane prior to staining for intracellularmarkers. Isotype matched control antibodies were treated under identicalconditions. Flow cytometric analysis was performed using a COULTER EPICSinstrument. The dot plots represent 5,000 listmode events indicating thelevel of fluorescence intensity for each lineage cell marker withreference to the isotype matched negative control antibodies.

Immunohistochemistry

Human tissue sections (μm) were de-waxed in xylene and rehydratedthrough graded ethanol into PBS. Frozen tissue sections (μm) andcytospin preparations were fixed with cold acetone at −20° C. for 15minutes then washed in PBS. The samples were subsequently treated withPBS containing 1.5% of hydrogen peroxide for 30 minutes, washed thenblocked with 5% non-immune goat serum for 1 hour at room . temperature.Samples were incubated with primary antibodies for 1 hour at roomtemperature. Antibodies used: Mouse (IgG₁ & IgG_(2a)) controls (Caltag,Burlingame, Calif.); Rabbit (Ig) control, 1A4 (anti-α smooth muscleactin, IgG₁), 2F11 (anti-neurofilament, IgG₁), F8/86 (murine anti-vonWillebrand Factor, IgG₁) (Dako, Carpinteria, Calif.); STRO-1; CC9(anti-CD146); LF-151 (rabbit anti-human dentinsialoprotein; Dr. L.Fisher, NIDCR/NIH, MD). Working dilutions: rabbit serum (1/500),monoclonal supernatants (1/2) and purified antibodies (10 μg/ml). Singlestaining was performed by incubating the samples with the appropriatesecondary antibody, biotinylated goat anti-mouse IgM, IgG₁, IgG_(2a) orbiotinylated goat anti-rabbit for one hour at room temperature (CaltagLaboratories). Avidin-Peroxidase-complex and substrate were then addedaccording to the manufacturer instructions (Vectastain ABC Kit standard,Vector Laboratories). Samples were counterstained with hematoxylin andmounted in aqueous media. Dual-fluorescence labeling was achieved byadding the secondary antibodies, goat anti-mouse IgM-Texas Red andIgG-FITC (CALTAG Laboratories), for 45 minutes at room temperature.After washing the samples were mounted in VECTASHIELD fluorescencemountant.

Immunomagnetic Bead Selection

Single cell suspensions of dental pulp tissue were incubated withantibodies reactive to STRO-1 (1/2), CD146 (1/2), or 3G5 (1/2) for 1hour on ice. The cells were washed twice with PBS/1% BSA then incubatedwith either sheep anti-mouse IgG-conjugated or rat anti-mouseIgM-conjugated magnetic Dynabeads (4 beads per cell: Dynal, Oslo,Norway) far 40 minutes on a rotary mixer at 4° C. Cells binding to beadswere removed using the MPC-1 magnetic particle concentrator (Dynal)following the manufactures recommended protocol.

Matrigel-Arteriole Assay

Single cell suspensions of ex vivo expanded bone marrow STRO-1^(bright)MPC were prepared by trypsin/EDTA treatment then plated into 48-wellplates containing 200 μl of matrigel. The STRO-1^(bright) MPC wereplated at 20,000 cells per well in serum-free medium (Gronthos et al.

2003) supplemented with the growth factors PDGF, EGF, VEGF at 10 ng/ml.Following 24 hours of culture at 37° C. in 5% CO₂, the wells were washedthen fixed with 4% paraformaldehyde. Immunohistochemical studies weresubsequently performed for alpha-smooth muscle actin identified with agoat-anti-murine IgG horse radish peroxidase antibody/Vectastaining Kitas described above.

Osteogenic, Adipogenic and Chondrogenic Differentiation of MPC in Vitro

Single cell suspensions of ex vivo expanded adipose-derived MPC werecultured in αMEM supplemented with 10% FCS, 100 μML-ascorbate-2-phosphate, dexamethasone 10⁻⁷ M and 3 mM inorganicphosphate previously shown to induce bone marrow MPC to form amineralized bone matrix in vitro (Gronthos et al., 2003). Mineraldeposits were identified by positive von Kossa staining. Adipogenesiswas induced in the presence of 0.5 mM methylisobutylmethylxanthine, 0.5μM hydrocortisone, and 60 μM indomethacin as previously described(Gronthos at al. 2003). Oil Red O staining was used to identifylipid-laden fat cells. Chondrogenic differentiation was assessed inaggregate cultures treated with 10 ng/ml TGF-β3 as described (Pittengeret al., 1999)

In Vivo Transplantation Studies

Approximately 5.0×10⁶ of ex vivo expanded cells derived from eitherSTRO-1^(bri)/CD146⁺ BMSSCs or CD146⁺ DPSCs were mixed with 40 mg ofhydroxyapatite/tricalcium phosphate (HA/TCP) ceramic powder (Zimmer Inc,Warsaw, Ind.) and then transplanted subcutaneously into the dorsalsurface of 10-week-old immunocompromised beige mice (NIH-bg-nu-xid,Harlan Sprague Dawley, Indianapolis, Ind.) as previously described.⁽⁴⁾These procedures were performed in accordance to specifications of anapproved animal protocol (NIDCR #00-113).

Reverse Transcription Polymerase Chain Reaction.

Total RNA was prepared from STRO-1^(BRT)/CD146⁺ sorted BMMNCs, andcontrol cells (primary BMSSC cultures grown in the presence of 10⁻⁷ Mdexamethasone for three weeks) using RNA STAT-60 (TEL-TEST Inc.Friendswood, Tex.). First-strand cDNA synthesis was performed with afirst-strand cDNA synthesis kit (GIBCO BRL, Life Technologies) using anoligo-dT primer. First strand cDNA (2 μl) was added to 46 μl of a 1× PCRmaster reaction mix (Roche Diagnostics, Gmbh Mannheim Germany) and 10pMol of each human specific primer sets: CBFAI (632 bp, and threesmaller alternative splice variants)⁽²⁷⁾ sense5′-CTATGGAGAGGACGCCACGCCTGG-3′, antisense, 5′-CATAGCCATCGTAGCCTBTCCT-3′;osteocalcin (310 bp)⁽⁴⁾ sense, 5′-CATGAGAGCCCTCACA-3′, antisense,5′-AGAGCGACACCCTAGAC-3′: GAPDH (800 bp)⁽⁴⁾ sense5′-AGCCGCATCTTCTITTGCGTC-3′; antisense 5′-TCATATTTGGCAGGTTTTTCT-3′. Thereactions were incubated in a PCR Express Hybaid thermal cycler (Hybaid,Franklin, Mass.) at 95° C. for 2 minutes for 1 cycle then 94°C./(30sec), 60° C./(30 sec), 72° C./(45 sec) for 35 cycles, with a final 7minute extension at 72° C. Following amplification, each reaction wasanalyzed by 1.5% agarose gel electrophoresis, and visualized by ethidiumbromide staining.

Results

BMSSCs and DPSCs Express Vascular Associated Antigens STRO-1 and CD146in Vivo.

We have previously demonstrated the efficacy of magnetic activated cellsorting (MACS), to isolate and enrich for all detectable clonogeniccolonies from aspirates of human marrow, based on their high expressionof STRO-1 antigen.^((25,26)) To further characterize BMSSCs we incubatedthe STRO-1^(bri) MACS isolated cells with another monoclonal antibody,CC9,⁽²⁸⁾ that recognizes the cell surface antigen CD146, also known asMUC-18, Mel-CAM and Sendo-1, that is present on endothelial and smoothmuscle cells. These studies determined that CC9, selectively bound theSTRO-1 bright expressing fraction (STRO-1^(BRT)) from the total STRO-1⁺population by dual-color FACS analysis (FIG. 10A). Cloning efficiencyassays using Poisson distribution statistics, yielded a marked increasein the incidence of BMSSCs (1 colony per 5 STRO-1^(BRT)/CD146⁺ cellsplated), and achieved a 2×10³ fold enrichment of the clonogenic colonypopulation when compared to unfractionated marrow (FIG. 10B). No colonyformation could be detected in STRO-1^(BRT)/CD146⁻ cell fraction (datanot shown).

The light scatter properties of STRO-1^(BRT)/CD146⁺ marrow cells weretypically larger and more granular than the nucleated erythroid cellsand B-lymphocytes comprising the bulk of the STRO-1⁺ population⁽²⁹⁾(FIG. 10C-E). Cytospin preparations of STRO-1^(BRT)/CD146⁺ sorted cellswere found to be negative for the erythroid (glycophorin-A) andleukocyte (CD45) associated markers (data not shown). Confirmation thatBMSSCs represented an early osteogenic precursor population was obtainedby RT-PCR analysis of highly purified MACS/FACS-isolated STRO-1^(BRT)/CD146⁺ cells, which failed to detect the early and late osteogenic,markers CBFA1 and osteocalcin, respectively (FIG. 10F). However, theprogeny of STRO-1^(BRT)/CD146⁺ sorted BMSSCs were found to express bothCBFA1 and osteocalcin, following ex vivo expansion. Immunolocalizationstudies demonstrated that the CD146 antigen was predominantly expressedon blood vessel walls in sections of human bone marrow (FIG. 10G).Localization of both STRO-1 and CD146 was confined to large bloodvessels in frozen sections of human bone marrow trephine (FIG. 10H).

Immunoselection protocols were subsequently used to determine if humanDPSCs also expressed STRO-1 and CD146 in situ. The use of either MACS orFACS analysis to isolate DPSCs was restrictive due to the rarity ofthese cells (1 colony-forming cell per 2×10³ cells plated) compounded bythe limited number of pulp cells (approximately 10⁵ cells per pulpsample) obtained following processing. To circumvent this, we pooledseveral pulp tissues obtained from 3 to 4 different third molars perexperiment and employed immunomagnetic bead selection on single cellsuspensions of pulp tissue, based on their expression of either theSTRO-1 or CD146 antigens. The STRO-1⁺ fraction represented approximately6% of the total pulp cell population. Comparative studies demonstratedthat growth rates of individual colonies were unperturbed in thepresence of magnetic beads (data not shown). Colony efficiency assaysindicated that the majority of dental pulp derived colony-forming cells(82%) were represented in the minor, STRO-1⁺ cell fraction analogous toBMSSCs (FIG. 11). The mean incidence of DPSCs in the STRO-1 positivefraction (329 colony-forming cells per 10⁵ cells plated±56 SE, n=3) wassix-fold greater than unfractionated pulp cells (55 colony-forming cellsper 10⁵ cells plated±14 SE, n=3). Using a similar strategy, differentfractions of human dental pulp cells were selected based on theirreactivity with the antibody, CC9. Colony efficiency assays showed thata high proportion (96%) of dental pulp-derived clonogenic colonies werealso present in the CD146⁺ population, using immunomagnetic Dynal beadselection (FIG. 11). The mean incidence of clonogenic colonies in theCD146⁺ fraction (296 colony-forming cells per 10⁵ cells plated±37 SE,n=3) was seven-fold greater than unfractionated pulp cells (42colony-forming cells per 10⁵ cells plated±9 SE, n=3).

Immunolocalization studies showed that STRO-1 expression was restrictedto blood vessel walls and perineurium surrounding the nerve bundles, butwas not present in the mature odontoblast layer or fibrous tissue, infrozen sections of human dental pulp tissue (FIG. 12A-B). Furthermore,co-localization of CD146 with STRO-1 was detected on the outer bloodvessel cell walls, with no reactivity to the surrounding fibrous tissue,odontoblast layer, and the perineurium of the nerve (FIG. 12C-D).Importantly, expression of human odontoblast-specific differentiationmarker, dentinsialoprotein (DSP), was restricted to the outer pulpallayer containing mature odontoblasts (FIG. 12E) and was absent infibrous tissue, nerve bundles and blood vessels.

Differential Expression of the Perivascular Marker 3G5 by BMSSCs andDPSCs.

In the present study, flow cytometric analysis revealed that the cellsurface antigen, 3G5, was highly expressed by a large proportion (54%)of hematopoietic marrow cells (FIG. 13A). This observation eliminated3G5 as a candidate marker for isolating purified populations of BMSSCsdirectly from aspirates of human marrow. In addition, dual-FACS analysisbased on 3G5 and STRO-1 expression was not possible since bothantibodies shared the same isotype. Nevertheless, in vitro colonyefficiency assays for different 3G5/CD146 FACS sorted subfractionsdemonstrated that only a minor proportion (14%) of bone marrowclonogenic colonies expressed the 3G5 antigen at low levels (FIG. 13B).Conversely, a larger proportion (63%) of clonogenic DPSCs (192colony-forming cells per 10⁵ cells plated±18.4 SE n=3) were present inthe 3G5⁺ cell fraction following immunomagnetic bead selection (FIG.11). 3G5 demonstrated specific reactivity to pericytes in frozensections of human dental pulp tissue (FIG. 12F).

We next analyzed the expression of more specific markers of endothelialcells (von Willebrand Factor) and smooth muscle cells/pericytes(α-smooth muscle actin) on cytospin preparations using freshly isolatedSTRO-1^(BRT)/CD146⁺ BMSSCs and CD146⁺ expressing DPSCs. A largeproportion of purified BMSSCs (67%), were found to be positive forα-smooth muscle actin (FIG. 14A), but lacked expression of vonWillebrand Factor (FIG. 14B). Similarly, the majority of isolated DPSCs(85%) were also found to express α-smooth muscle actin, but not vonWillebrand Factor (FIG. 14C, 5D). Purified populations ofSTRO-1^(BRT)/CD146⁺ BMSSCs and CD146⁺ DPSCs were subsequently expandedin vitro then transplanted into immunocompromised mice to assess theirdevelopmental potentials in vivo. The progeny of cultured BMSSCs andDPSCs displayed distinct capacities, capable of regenerating the bonemarrow and dental/pulp microenvironments, respectively (FIG. 14E, F),and appeared identical to the developmental potential of non-selectedmulti-colony derived BMSSCs and DPSCs (4).

Discussion

The present study provides direct evidence that two mesenchymal stemcell populations, distinct in their ontogeny and developmentalpotentials, are both associated with the microvasculature of theirrespective tissues.

We employed different immunoselection protocols to demonstrate thatBMSSCs and DPSCs could be efficiently retrieved from bone marrowaspirates and enzyme digested pulp tissue respectively, based primarilyon their high expression of the STRO-1 antigen. This cell surfaceantigen is present on precursors of various stromal cell typesincluding, marrow fibroblasts, osteoblasts, chondrocytes, adipocytes,and smooth muscle cells isolated from human adult and fetal bonemarrow.^((29,32-34)) Previous studies have implicated STRO-1 as a markerof pre-osteogenic populations, where its expression is progressivelylost following cell proliferation and differentiation into matureosteoblasts in vitro.^((27,35,36)) The STRO-1 antigen was also found tobe present on the outer cell walls of human bone marrow and dental pulpblood vessels, in accord with previous studies that localized STRO-1 onlarge blood vessels, but not capillaries, in different adult tissuessuch as brain, gut, heart, kidney, liver, lung, lymphnode, muscle,thymus.⁽⁶⁾ Therefore, STRO-1 appears to be an early marker of differentmesenchymal stem cell populations and infers a possible perivascularniche for these stem cell populations in situ.

To determine if BMSSCs and DPSCs were associated directly with bloodvessels we utilized another antibody (CC9),⁽²⁸⁾ which recognizes theimmunoglobulin super family member, CD146 (MUC-18/Mel-CAM), known to bepresent on smooth muscle, endothelium, myofibroblasts and Schwann cellsin situ, as well as being a marker for some human neoplasms.⁽³⁷⁾Notably, CD146 is not expressed by bone marrow haematopoietic stemcells, nor their progenitors. While the precise function of CD146 is notknown, it has been linked to various cellular processes including celladhesion, cytoskeletal reorganization, cell shape, migration andproliferation through transmembrane signaling.

In order to dissect the BMSSC population, STRO-1^(BRT) expressing marrowcells were further distinguished from STRO-1⁺ haematopoietic cells(predominantly glycophorin-A⁺ nucleated erythrocytes) based on theirexpression of CD146, using dual-FACS analysis. PurifiedSTRO-1^(BRT)/CD146⁺ human BMSSCs displayed light scatter propertiescharacteristic of large granular cells. Our study supports the findingsof Van Vlasselaer and colleagues (1994)⁽³⁸⁾ who isolated partiallypurified BMSSCs from murine bone marrow following 5-fluoracil (5-FU)treatment, and identified this population as having high perpendicularand forward light scatter characteristics. Interestingly, freshlyisolated 5-FU resistant murine BMSSCs were also found to be positive fortwo perivascular markers Sab-1 and Sab-2.⁽³⁸⁾ Conversely, more recentstudies have shown that when BMSSCs are cultivated in vitro, the mostprimitive populations display low perpendicular and forward lightscatter properties⁽³⁹⁾ and therefore may not reflect the true morphologyof BMSSC in situ. In the present study, STRO-1^(BRT)/CD146⁺ sorted humanBMSSCs lacked the expression of CBFAI and osteocalcin that identifycommitted early and late osteogenic populations, respectively,^((40,41))indicating that BMSSCs exhibit a pre-osteogenic phenotype in human bonemarrow aspirates. We found that a high proportion of freshly isolatedSTRO-1^(BRT)/CD146⁺ BMSSCs expressed α-smooth muscle actin, but not theendothelial specific marker von Willebrand Factor, providing directevidence that this primitive precursor population displays acharacteristic perivascular phenotype.

The present study also demonstrated the efficacy of using magnetic beadselection to isolate and enrich for DPSCs directly from human dentalpulp tissue based on their expression of either STRO-1 or CD146.Immunolocalization of CD146 appeared to be specific to themicrovasculature within dental pup. Co-localization of both STRO-1 andCD 146 on the outer walls of large blood vessel in dental pulp tissue,implied that the majority of DPSCs arise from the microvasculature.However, since the STRO-1 antibody also reacted with the perineurium indental pulp and peripheral nerve bundles (unpublished observations),further investigation is required to determine the role of this antigenin neural cell development.

Analogous to BMSSCs, freshly isolated CD146⁺ DPSCs were found to expressα-smooth muscle actin but not von Willebrand Factor. DPSCs were alsoshown to be an immature pre-odontogenic population both by theirlocation distal from the dentin forming surface and by their lack ofexpression of the human odontoblast-specific dentin sialoprotein (DSP),which is restricted to the outer pulpal layer containing differentiatedodontoblasts. We have previously described that ex vivo expanded humanDPSCs do not express the precursor molecule, dentinsialophosphoprotein(DSPP), in vitro when cultured under non-inductive conditions.⁽⁴⁾Similar studies have shown that DSPP mRNA was highly expressed infreshly isolated odontoblast/pulp tissue, but was not detect in cultureddental papilla cells derived from rat incisors.^((43,44)) It is onlywhen DPSCs are induced, either in vitro,⁽⁴⁵⁾ or by in vivotransplantation to form an ordered dentin matrix that DSPP isexpressed.⁽⁴⁾

In vitro studies of ex vivo expanded BMSSCs and DPSCs supported thenotion that their progeny were morphologically similar to culturedperivascular cells having a bi-polar fibroblastic, stellar or flatmorphology, rather than a polygonal endothelial-like appearance. Inaddition, we have previously shown that the progeny of BMSSC- andDPSC-derived colonies exhibit heterogeneous staining for both CD146 andα-smooth muscle actin, but lack expression of the endothelial markers,CD34 and von Willebrand Factor, in vitro.⁽⁴⁾

The observations that two different mesenchymal stem cell populationssuch as BMSSCs and DPSCs harbour in perivascular niches may have furtherimplications for identifying stem cell populations in other adulttissues. Recent findings have identified human “reserve” multi-potentmesenchymal stem cells in connective tissues of skeletal muscle, anddermis derived from human fetal and adult samples.⁽⁵⁶⁾ However the exactlocation, developmental potential and ontogeny of these stem cells isstill largely unknown. In the present study, identification ofmesenchymal stem cell niches in bone marrow and dentin pulp may helpelucidate the fundamental conditions necessary to selectively maintainand expand primitive multi-potential populations in vitro, in order todirect their developmental potentials in vivo.

EXAMPLE 8 Adult Human Bone Marrow MPC are Distinct from StromalPrecursor Cells, Haematopoietic Stem Cells and Angioblasts by Their HighExpression of the STRO-1 Antigen and Lack of CD34 Expression

Postnatal bone marrow appears to be a hub of residential stem andprecursor cell types responsible for blood cell formation(haematopoietic stem cells), endothelial development (angioblast), andconnective tissue/stromal differentiation (stromal precursor cells/bonemarrow stromal stem cells/mesenchymal stem cells). Recent work by ourgroup (Gronthos et al. 2003; Shi and Gronthos 2003) has, for the firsttime, purified and characterised human multipotential bone marrowmesenchymal precursor cells (MPC) based on their high expression of theSTRO-1 antigen and by their co-expression of the immunoglobulinsuperfamily members, VCAM-1 (CD106) and MUC-18 (CD146). Early studies bySimmons and Torok-Storb (1991a and b), have shown that bonemarrow-derived STRO-1⁺ stromal precursor cells, with the capacity toform adherent colonies in vitro, also expressed the haematopoietic stemcell marker, CD34, albeit at low levels. These studies used CD34antibody-complement mediated cell lysis to eliminate a high proportionof adherent colony-forming cells in marrow aspirates (Simmons andTorok-Storb 1991b). It is important to note that while the STRO-1antibody was generated following immunisation of mice with human CD34⁺bone marrow cells, this may have arisen due to the fact that the STRO-1antigen is also expressed at moderate to low levels onCD34⁺/Glycophorin-A⁺ nucleated red cells and CD34⁺/CD20⁺ B-lymphocytes.We now offer direct evidence, using sophisticated fluorescence activatedcell sorting technology that multipotential adult human bone marrow MPCexpress high levels of STRO-1, but lack expression to the stromalprecursor cell, haematopoietic stem cell and angioblast maker (CD34),the leukocyte antigen (CD45), and the nucleated red cell marker(Glycophorin-A) (FIG. 15A-C). These data demonstrate that adult humanbone marrow-derived MPC are a novel stem cell population, distinct frommore mature stromal precursor cells, haematopoietic stem cells andangioblast (FIG. 16).

Unless otherwise indicated the materials and methods of this example arethe same as those for Example 1.

FIG. 15. Expression of CD34, CD45 and Glycophorin-A on STRO-1 positivebone marrow mononuclear cells. Representative histograms depictingtypical dual-colour flow cytometric analysis profiles of STRO-1 positivebone marrow mononuclear cells isolated initially by magnetic activatedsorting and co-stained with antibodies directed against CD34 (A), CD45(B) or Glycophorin-A (C). The STRO-1 antibody was identified using agoat anti-murine IgM-fluorescein isothiocyanate while CD34, CD45 andGlycophorin-A were identified using a goat anti-murineIgG-phycoerythrin. The high expressing STRO-1 fraction which containedthe clonogenic MPC population was isolated by fluorescence activatedcell sorting based on regions R1 and R2.

FIG. 16. Bone marrow MPC are STRO-1 bright, CD34 negative, CD45 negativeand Glycophorin-A negative. The graph depicts the results of in vitroadherent colony formation assays performed for each of the differentsorted STRO-1 bright populations selected by their co-expression or lackof either the CD34, CD45 or Gycophorin-A antigens, based on regions R1and R2 as indicated in FIG. 15. These data are expressed as the meanincidence of colony-forming units for each cell fraction averaged fromtwo separate experiments.

EXAMPLE 9 Identification of Multipotential MPC in Different HumanTissues

While the existence and precise location of MPC in different tissues islargely unknown, we have recently demonstrated that MPC appear to residein a perivascular niche in human bone marrow and dental pulp tissues(Shi and Gronthos 2003). These observations were based on a combinationof immunohistochemical and immunoselection methods to identify andisolate different MPC populations based on their expression of themesenchymal stem cell marker, STRO-1, the smooth muscle and pericytemarkers, CD146, alpha-smooth muscle actin and the pericyte specificmarker, 3G5. We have now extended these studies demonstrating theco-localization of STRO-1/CD146, STRO-1/alpha-smooth muscle actin, and3G5/CD146 antigens in a wider variety of tissues including heart, liver,kidney, skin, spleen, pancreas, lymph node (FIG. 17).

To confirm our earlier findings that MPC can be derived from non-bonemarrow tissue such as dental pulp, we used fluorescence activated cellsorting to isolate different MPC populations from adult human peripheraladipose. Single cell suspensions were obtained following digestion ofthe adipose tissue with collagenase and dispase as previously described(Shi and Gronthos 2003). The adipose-derived cells were then incubatedwith antibodies reactive against STRO-1, CD146 and 3G5. Cell populationswere then selected by FACS, based on their positivity (region R3) ornegativity (region R2) to each marker and then plated into regulargrowth medium (Shi and Gronthos 2003) to assess the incidence ofadherent colony-forming cells in each cell fraction (FIG. 18). Following12 days of culture, colonies (aggregates of 50 cells or more) werescored and displayed as the number of colonies per 10⁵ cells plated foreach cell fraction. Our data demonstrated that MPC can be derived fromadipose tissues based on their expression of STRO-1/3G5/CD146 antigens(FIG. 19). Dual colour flow cytometric analysis confirmed that only aminor proportion of adipose-derived cells co-expressed STRO-1/CD146 and3G5/CD146 (FIG. 20). These findings are consistent with our previousobservations that MPC can be isolated from both bone marrow and dentalpulp tissue based on the same set of perivascular markers (Shi andGronthos 2003). Furthermore, we provide evidence demonstrating thatadipose derived MPC isolated by CD146 selection have the capacity todifferentiate into different tissues such as bone, fat and cartilage(FIG. 21), as previous described (Gronthos et al. 2003).

Recent findings examining the existence of MPC in unrelated tissues suchas skin has also been examined to further strengthen our hypothesis.Single cell suspensions were obtained following digestion of fullthickness human skin with collagenase and dispase as described above forhuman adipose tissue. The skin-derived cells were then incubated withantibodies reactive against STRO-1, CD146 and 3G5 identified usingeither a goat anti-murine IgM or IgG-phycoerythrin. Cell populationswere then selected by FACS, based on their positivity (region R3) ornegativity (region R2) to each marker and then plated into regulargrowth medium (Shi and Gronthos 2003) to assess the incidence ofadherent colony-forming cells in each cell fraction (FIG. 22). Following12 days of culture, colonies (aggregates of 50 cells or more) werescored and displayed as the number of colonies per 10⁵ cells plated foreach cell fraction. The data demonstrated that MPC can also be derivedfrom skin based on their expression of STRO-1/3G5/CD146 antigens (FIG.19). Collectively these data suggest that multipotential MPC can beidentified and isolated in virtually all vascularised tissues derivedfrom postnatal human tissue based on a common phenotype.

Unless otherwise indicated the materials and methods of this example arethe same as those for Example 1.

FIG. 17. Reactivity of perivascular makers in different human tissues.Dual-colour immunofluorescence staining demonstrating reactivity of (A)STRO-1 and CD146, (B) STRO-1 and alpha-smooth muscle actin, and (C) 3G5and CD146, on blood vessels and connective tissue present on spleen,pancreas (Panel 1), brain, kidney (Panel II), liver, heart (Panel III)and skin (Panel IV) 20×. The STRO-1 and 3G5 antibodies were identifiedusing a goat anti-murine IgM-Texas Red while CD146 and alpha-smoothmuscle actin were identified using a goat anti-murine or IgG-fluoresceinisothiocyanate. Co-localization is indicated by overlapping areas ofyellow and orange fluorescence (white arrows),

FIG. 18. Isolation of adipose-derived MPC by FACS. Representative flowcytometric histograms depicting the expression of STRO-1, CD146 and 3G5in fresh preparations of peripheral adipose-derived single-cellsuspensions generated following collagenase/dispase digestion aspreviously described (Shi and Gronthos 2003). The antibodies wereidentified using either a goat anti-murine IgM or IgG-phycoerythrin.Cell populations were then selected by FACS, based on their positivity(region R3) or negativity (region R2) to each marker and then platedinto regular growth medium to assess the incidence of adherentcolony-forming cells in each cell fraction.

FIG. 19. Clonogenic adipose-derived MPC are positive forSTRO-1/3G5/CD146. The bar graph depicts the number of clonogeniccolonies retrieved from single cell suspensions of enzymaticallydigested human peripheral adipose tissue, following fluorescenceactivated cell sorting, based on their reactivity to antibodies thatrecognize STRO-1, CD146, and 3G5 (FIG. 18), then cultured in standardgrowth medium as previously described for bone marrow and dental pulptissue (Shi and Gronthos 2003). The data are expressed as the number ofcolony-forming units obtained per 10³ cells plated in the positive andnegative cell fractions averaged from two separate experiments.

FIG. 20. Immunophenotypic analysis of adipose-derived MPC.Representative flow cytometric histograms depicting the co-expression ofSTRO-1 and CD146 (A) and 3G5 and CD146 in fresh preparations ofperipheral adipose-derived single-cell suspensions generated followingcollagenase/dispase digestion. The STRO-1 and 3G5 antibodies wereidentified using a goat anti-murine IgM-phycoerythrin while CD146 wasidentified using a goat anti-murine IgG-fluorescein isothiocyanate.

Approximately 60% and 50% of the CD146 positive cells co-express STRO-1and 3G5, respectively. These data suggest that 10% or more of the CD164positive cells co-express STRO-1 and 3G5.

FIG. 21. Developmental potential of purified Adipocyte-derived MPC invitro. Preparations of primary MPC cultures derived from STRO-1⁺/CD146⁺adipose cells were re-cultured either in standard culture conditions(A), osteogenic inductive medium (B), Adipogenic inductive medium (C) orcondrogenic conditions (D) as previously described Gronthos et al. 2003.Following two weeks of multi-differentiation induction, theadipocyte-derived MPC demonstrated the capacity to form bone (B;Alizarin positive mineral deposits), fat (C; Oil Red O positive lipid)and cartilage (D: collagen type U matrix).

FIG. 22. Isolation of skin-derived MPC by FACS. Representative flowcytometric histograms depicting the expression of STRO-1, CD146 and 3G5in fresh preparations of full thickness skin-derived single-cellsuspensions generated following collagenase/dispase digestion. Theantibodies were identified using either a goat anti-murine IgM orIgG-phycoerythrin. Cell populations were then selected by FACS, based ontheir positivity (region R3) or negativity (region R2) to each markerand then plated into regular growth medium to assess the incidence ofadherent colony-forming cells in each cell fraction.

FIG. 23. Clonogenic skin-derived MPC are positive for STRO-1bri/3G5/CD146. The bar graph depicts the number of adherent coloniesrecovered from single cell suspensions of enzymatically digested humanskin, following fluorescence activated cell sorting, based on theirreactivity to antibodies that recognize STRO-1, CD146, and 3G5, thencultured in standard growth medium as previously described for bonemarrow and dental pulp tissue (Shi and Gronthos 2003). The data areexpressed as the number of colony-forming units obtained per 10⁵ cellsplated in the positive and negative cell fractions averaged from twoseparate experiments.

EXAMPLE 10 l Immunophenotypic Analysis of Ex Vivo Expanded Human BoneMarrow Mesenchymal Precursor Cells

We have previously reported that multipotential mesenchymal precursorcells (MPC) can be purified from adult human bone marrow mononuclearcells based on the phenotype STRO-1^(bright)/VCAM-1 (CD106)⁺ orSTRO-1^(bright)/MUC-18 (CD146)⁺ (Gronthos et al. 2003; Shi and Gronthos2003). The MPC population can be readily propagated in vitro underdefined culture conditions (Gronthos et al. 2003). We now present datacharacterising the ex vivo expanded MPC progeny based on markersassociated with different cell lineages, at both the mRNA and proteinlevel, using reverse transcriptase-polymerase chain reaction (RT-PCR)and flow cytometric analysis, respectively.

In the first series of experiments, semi-quantitative RT-PCR analysiswas employed to examine the gene expression profile of variouslineage-associated genes present in the cultured MPC populations (FIG.24). Relative gene expression for each cell marker was assessed withreference to the expression of the house-keeping gene, GAPDH, usingImageQuant software (FIG. 24B). In addition, single-colour flowcytometric analysis was used to examine the protein expression profileof ex vivo expanded MPC based on their expression of celllineage-associated markers (FIG. 24A). A summary of the generalphenotype based on the gene and protein expression of the cultured MPCis presented in Table 1. Direct comparison of the gene expressionprofile of MPC described in the present patent demonstrated cleardifferences between this cell population and mesenchymal stem cells(MSC) previously described by Pittenger et al. 1999, (Table 1).

Unless otherwise indicated the materials and methods of this example arethe same as those for Example 1.

FIG. 24A. Immunophenotypic expression pattern of ex vivo expanded bonemarrow MPC. Single cell suspensions of ex vivo expanded bone marrow MPCwere prepared by trypsin/EDTA treatment then incubated with antibodiesidentifying cell lineage-associated markers. For those antibodiesidentifying intracellular antigens, cell preparations were fixed withcold 70% ethanol to permeanbilize the cellular membrane prior tostaining for intracellular markers. Isotype matched control antibodieswere treated under identical conditions. Flow cytometric analysis wasperformed using a COULTER EPICS instrument. The dot plots represent5,000 listmode events indicating the level of fluorescence intensity foreach lineage cell marker (bold line) with reference to the isotypematched negative control antibodies (thin line).

FIG. 24B. Gene expression profile of cultured MPC. Single cellsuspensions of ex vivo expanded bone marrow MPC were prepared bytrypsin/EDTA treatment and total cellular RNA was prepared. UsingRNAzolB extraction method total RNA was isolated and used as a templatefor cDNA synthesis, prepared using standard procedure. The expression ofvarious transcripts was assessed by PCR amplification, using a standardprotocol as described previously (Gronthos et al. 2003). Primers setsused in this study are shown in Table 2. Following amplification, eachreaction mixture was analysed by 1.5% agarose gel electrophoresis, andvisualised by ethidium bromide staining. Relative gene expression foreach cell marker was assessed with reference to the expression of thehouse-keeping gene, GAPDH, using ImageQuant software.

FIG. 25. Ex vivo expanded STRO-1 MPC can develop into arterioles invitro. Single cell suspensions of ex vivo expanded bone marrowSTRO-1^(bri) and STRO-1^(dull) MPC were prepared by trypsin/EDTAtreatment then plated into 48-well plates containing 200 μl of matrigel.The STRO-1^(dull) (A) and STRO-1^(bri) (B) MPC were plated at 20,000cells per well in serum-free medium (Gronthos et al. 2003) supplementedwith the growth factors PDGF, EGF, VEGF at 10 ng/ml. Following 24 hoursof culture at 37° C. in 5% CO₂, the wells were washed then fixed with 4%paraformaldehyde. Immunohistochemical studies were subsequentlyperformed demonstrated that the cord-like structures expressedalpha-smooth muscle actin identified with a goat-anti-murine IgG horseradish peroxidase antibody.

TABLE 1 Comparison between cultured human Mesenchymal Precursor Cells(MCP's) and cultured human Mesenchymal Stem Cells (MSC's) following exvivo expansion. Differentiated ANTIGEN MSC MPC Cell Type. STRO-1 −ve +veCollagen II −ve +ve Chondrocyte (MES) Collagen IV −ve +ve Fibroblast(MES) Laminin −ve +ve Fibroblast (MES) Bone Sialoprotein −ve +veOsteoblast (MES) (BSP) Osteocalcin (OCN) −ve +ve Osteoblast (MES) NestinND +ve Neural (ECT) Glial Fibrillary Acidic ND +ve Neural (ECT) Protein(GFAP) CBFA1 −ve +ve Osteoblast (MES) Osterix (OSX) ND +ve Osteoblast(MES) Osteocalcin (OCN) −ve +ve Osteoblast (MES) Sox9 ND +ve Chondrocyte(MES) Collagen X (COL X) +ve +ve Chondrocyte (MES) Leptin ND +ve Adipose(MES) GATA-4 ND +ve Cardiomyocyte (MES) Transferrin (TFN) ND +veHepatocyte (END) Flavin Containing ND +ve Hepatocyte (END) Monooxygenase(FCM) Antigens found to be present on cell surface, intracellular or inthe extra cellular matrix. MPCs express markers of tissues withdifferent developmental origin, ie. ECT—ectoderm, MES—mesoderm andEND—endoderm.

TABLE 2 RT-PCR primers and conditions for the specificamplification of human mRNA Target GeneSense/Antisense (5′-3′) Primer Sequences Product Size GAPDHCACTGACACGTTGGCAGTGG/ 417 CATGGAGAAGGCTGGGGCTC LectinATGCATTGGGAACCCTGTGC/ 492 GCACCCAGGGCTGAGGTCCA CBFA-1GTGGACGAGGCAAGAGTTTCA/ 632 TGGCAGGTAGGTGTGGTAGTG OCNATGAGAGCCCTCACACTCCTC/ 289 CGTAGAAGCGCCGATAGGC GFAPCTGTTGCCAGAGATGGAGGTT/ 370 TCATCGCTCAGGAGGTCCTT NestinGGCAGCGTTGGAACAGAGGTTGGA/ 460 CTCTAAACTGGAGTGGTCAGGGCT GATA-4GACTTCTCAGAAGGCAGAG/ 800 CTATCCTCCAAGTCCCAGAG PDGFβ-RAATGTCTCCAGCACCTTCGT/ 650 AGCGGATGTGGTAAGGCATA OsterixGGCACAAAGAAGCCGTACTC/ 247 CACTGGGCAGACAGTCAGAA COL XAGCCAGGGTTGCCAGGACCA/ 387 TTTTCCCACTCCAGGAGGGC SOX9CTC TGC CTG TTT GGA CTT TGT/ 598 CCT TTG CTT GCC TTT TAC CTC Ang-1CCAGTCAGAGGCAGTACATGCTA AGAATTGAGTTA/ 300 GTTTTCCATGGTTTTGTCCCGCAGTA

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1-67. (canceled)
 68. A process for making a population of mesenchymalprecursor cells comprising obtaining a population of cells comprisingcells which express STRO-1 at a higher level and cells which expressSTRO-1 at a lower level, relative to each other, and enriching thepopulation for cells which express STRO-1 at the higher level relativeto cells which express STRO-1 at the lower level, thereby making thepopulation of mesenchymal precursor cells.
 69. The process of claim 68,wherein the cells which express STRO-1 at the higher level also expressat least one other antigen selected from the group consisting of LFA-3,ICAM-1, PECAM-1, P-selectin, L-selectin, CD49b/CD29, CD49c/CD29,CD49d/CD29, CD29, CD18, CD61, 6-19, thrombomodulin, telomerase, CD10,CD13, integrin beta, and SCF.
 70. The process of claim 68, wherein theenriching step is performed so as to make a cell population in which atleast 1% of the cells are capable of giving rise to colony formingunits-fibroblast (CFU-F).
 71. The process of claim 68, wherein theenriching step is performed so as to make a cell population in which atleast 5% of the cells are capable of giving rise to colony formingunits-fibroblast (CFU-F).
 72. The process of claim 68, wherein theenriching step is performed so as to make a cell population in which atleast 10% of the cells are capable of giving rise to colony formingunits-fibroblast (CFU-F).
 73. The process of claim 68, wherein theenriching step is performed so as to make a cell population in which atleast 40% of the cells are capable of giving rise to colony formingunits-fibroblast (CFU-F).
 74. The process of claim 68, wherein theenriching step is performed so as to make a cell population in which atleast 2% of the cells carry both the antigen identified by STRO-1 andVCAM-1.
 75. The process of claim 68, wherein the enriching step isperformed so as to make a cell population in which cells which expressSTRO-1 at the higher level are negative for at least one marker selectedfrom the group consisting of CBFA-1, collagen type II, PPARγ2,osteopontin, osteocalcin, parathyroid hormone receptor, leptin, humanadipocyte lipid binding protein (H-ALBP), aggrecam, Ki67, andglycophorin A.
 76. The process of claim 68, wherein the enriching stepis performed so as to make a cell population in which at least 1.5% ofthe cells in the population express STRO-1 at the higher level.
 77. Theprocess of claim 68, wherein the enriching step is performed so as tomake a cell population in which at least 2% of the cells in thepopulation express STRO-1 at the higher level.
 78. The process of claim68, wherein the population of cells is stromal stem cells obtained frombone marrow, blood, epidermis, or hair follicles.
 79. The process ofclaim 78, wherein the population of cells is obtained from bone marrow.80. The process of claim 68, wherein obtaining the population of cellscomprises harvesting the population of the cells from a subject.
 81. Theprocess of claim 68, wherein the cells which express STRO-1 at thehigher level also express at least one other marker selected fromVCAM-1, THY-1, CD146, and STRO-2.
 82. The process of claim 68, whereinthe enriching step comprises a separation step based on recognition ofcells expressing the STRO-1 marker.
 83. The process of claim 82, whereinthe recognition of cells expressing the STRO-1 marker is achieved by abinding agent which binds to a marker on the surface of the cellsexpressing the STRO-1 marker.
 84. The process of claim 83, wherein thebinding agent is a monoclonal antibody or molecule based on a monoclonalantibody.
 85. The process of claim 68, wherein at least a proportion ofthe population of mesenchymal precursor cells is capable ofdifferentiation into at least two committed cell types each selectedfrom the group consisting of adipose, areolar, osseous, cartilaginous,elastic, and fibrous connective tissue.
 86. The process of claim 68,further comprising transforming the cell population with an exogenousnucleic acid.